U.S. patent number 11,072,798 [Application Number 16/570,489] was granted by the patent office on 2021-07-27 for transcriptional regulation for improved plant productivity.
This patent grant is currently assigned to YIELD 10 BIOSCIENCE, INC.. The grantee listed for this patent is YIELD10 BIOSCIENCE, INC.. Invention is credited to Madana M. R. Ambavaram, Mariya Somleva.
United States Patent |
11,072,798 |
Ambavaram , et al. |
July 27, 2021 |
Transcriptional regulation for improved plant productivity
Abstract
Methods comprising DNA constructs and polynucleotides of
functional transcription factors for improving photosynthetic
capacity, biomass and/or grain yield and stress tolerance in
various crop and model plants, dicots and monocots with the C.sub.3
or C.sub.4 photosynthetic pathways are described herein.
Inventors: |
Ambavaram; Madana M. R.
(Norwood, MA), Somleva; Mariya (Cambridge, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
YIELD10 BIOSCIENCE, INC. |
Woburn |
MA |
US |
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Assignee: |
YIELD 10 BIOSCIENCE, INC.
(Woburn, MA)
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Family
ID: |
1000005699660 |
Appl.
No.: |
16/570,489 |
Filed: |
September 13, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200048651 A1 |
Feb 13, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15897958 |
Feb 15, 2018 |
10450580 |
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14653431 |
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PCT/US2013/076308 |
Dec 18, 2013 |
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61738675 |
Dec 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/8271 (20130101); C12N 15/8261 (20130101); C12N
15/8273 (20130101); C12N 15/8251 (20130101); C07K
14/415 (20130101); C12N 15/8245 (20130101); C12N
15/8241 (20130101); Y02A 40/146 (20180101) |
Current International
Class: |
C12N
15/82 (20060101); C07K 14/415 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2005/112608 |
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Dec 2005 |
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WO |
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2014/093614 |
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Jun 2014 |
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WO |
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Other References
Abate et al., "Separation and structural characterization of cyclic
and open chain oligomers produced in the partial pyrolysis of
microbial poly(hydroxybutyrates)," Macromolecules, 28(23):
7911-7916 (1995). cited by applicant .
Dietz et al., "AP2/EREBP transcription factors are part of gene
regulatory networks and integrate metabolic, hormonal and
environmental signs in stress acclimation and retrograde
signalling," Protoplasma, 245(1-4): 3-14 (2010). cited by applicant
.
International Search Report and Written Opinion for International
Application No. PCT/US2013/076308 dated Jun. 4, 2014 (our reference
MBQ-01125). cited by applicant .
Invitation to Pay Additional Fees for PCT/US2013/076308,
"Transcriptional Regulation for Improved Plant Productivity;" dated
Mar. 27, 2014. (4614.1011-001). cited by applicant .
Jaglo et al., "Components of the Arabidopsis
C-Repeat/Dehydration-Responsive Element Binding Factor
Cold-Response Pathway are Conserved in Brassica napus and Other
Plant Species," Plant Physiology, 127: 910-917 (Nov. 2001). cited
by applicant .
Mizoi et al., "AP2/ERF family transcription factors in plant
abiotic stress responses," Biochim Biophys Acta., 1819(2): 86-96
(2012). cited by applicant .
Nakano et al., "Genome-Wide Analysis of the ERF Gene Family in
Arabidopsis and Rice," Plant Physiology, 140: 411-432 (Feb. 2006).
cited by applicant .
Notification of Transmittal of the International Search Report and
the Written Opinion of the International Search Authority for
PCT/US2013/076308, "Transcriptional Regulation for Improved Plant
Productivity;" dated Jun. 4, 2014.(4614.1011-001). cited by
applicant .
Ohto et al., "Control of seed mass by APETALA2," PNAS, 102(8):
3123-3128 (2005). cited by applicant .
Saibo et al., "Transcription factors and regulation of
photosynthetic and related metabolism under environmental
stresses," Annals of Botany, 103(4): 609-623 (2009). cited by
applicant .
Zhang et al., "Overexpression of the Soybean GmERF3 Gene, a AP2/ERF
Type Transcription Factor for Increased Tolerances to Salt,
Drought, and Diseases in Transgenic Tobacco," J Exp Botany, 60(13):
3781-3796 (2009). cited by applicant .
Zhang et al., "Progresses on Plant AP2/ERF Transcription Factors,"
Hereditas, 34(7): 835-847 (2012). cited by applicant.
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Primary Examiner: Buran; Ashley K
Attorney, Agent or Firm: Pearne & Gordon LLP
Government Interests
GOVERNMENT SUPPORT
This invention was made with government support under Award Number
DE-EE0004943 awarded by Department of Energy. The government has
certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
15/897,958, filed Feb. 15, 2018, which is a continuation of U.S.
application Ser. No. 14/653,431, filed Jun. 18, 2015, which is the
U.S. National Stage of International Application No.
PCT/US2013/076308, filed on Dec. 18, 2013, published in English,
which claims the benefit of U.S. Provisional Application No.
61/738,675, filed on Dec. 18, 2012, all of which are hereby
incorporated by reference.
Claims
What is claimed is:
1. A method for producing a genetically modified plant or plant
cell having an increased carbon flow, the method comprising: a)
transforming one or more host plants or plant cells with a vector
comprising a nucleic acid encoding a polypeptide having at least
95% identity to SEQ ID NO: 4 operably linked to a promoter; and (b)
selecting one or more transformed plants or plant cells for
increased carbon flow by selecting for one or more of increased
biomass yield, increased starch yield, increased glucose content,
or increased sucrose content as compared to a control plant lacking
the vector, wherein the promoter is light inducible.
2. The method according to claim 1, the method further comprising
collecting seeds comprising the vector from the selected plant.
3. The method according to claim 1, the method further comprising
regenerating a plant from the selected plant cell and collecting
seeds comprising the vector from the regenerated plant.
4. The method of claim 1, wherein the selected plant further
exhibits increased tolerance to one or more abiotic stress factors
of excess or deficiency of water and/or light, high or low
temperature, or high salinity, as compared to a control plant
lacking the vector.
5. The method of claim 1, wherein the promoter comprises positions
8951 to 10645 of SEQ ID NO: 21.
6. The method of claim 1, wherein the host plant is a
monocotyledonous plant or a dicotyledonous plant.
7. The method of claim 6, wherein the host plant is
switchgrass.
8. The method of claim 6, wherein the host plant is maize.
9. The method of claim 6, wherein the host plant is sugar cane.
10. The method according to claim 1, wherein the host plant is
Miscanthus, Medicago, sweet sorghum, grain sorghum, sugar cane,
energy cane, elephant grass, maize, wheat, barley, oat, rice,
soybean, oil palm, safflower, sesame, flax, cotton, sunflower,
Camelina, Brassica napus, Brassica carinata, Brassica juncea, pearl
millet, or foxtail millet.
11. The method of claim 1, wherein the polypeptide has at least 99%
sequence identity to SEQ ID NO: 4.
12. The method of claim 11, wherein the host plant is Miscanthus,
Medicago, sweet sorghum, grain sorghum, sugar cane, energy cane,
elephant grass, maize, wheat, barley, oat, rice, soybean, oil palm,
safflower, sesame, flax, cotton, sunflower, Camelina, Brassica
napus, Brassica carinata, Brassica juncea, pearl millet, or foxtail
millet.
13. The method of claim 1, wherein the polypeptide comprises SEQ ID
NO: 4.
14. The method of claim 13, wherein the host plant is Miscanthus,
Medicago, sweet sorghum, grain sorghum, sugar cane, energy cane,
elephant grass, maize, wheat, barley, oat, rice, soybean, oil palm,
safflower, sesame, flax, cotton, sunflower, Camelina, Brassica
napus, Brassica carinata, Brassica juncea, pearl millet, or foxtail
millet.
15. The method according to claim 1, wherein the nucleic acid
comprises SEQ ID NO: 1.
Description
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The material in the ASCII text file, named
"YTEN-60124US3-Sequence-Listing_ST25.txt", created Sep. 9, 2019,
file size of 180,224 bytes, is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
The increasing size of the global population, the increasing
standard of living in emerging nations such as China and the use of
renewable resources such as plants to produce biofuels and
bio-based chemicals has placed additional pressure on agriculture.
These factors together with the limited availability of additional
arable land and water resources means that crop productivity or
yield is the key to feeding these demands. Agriculture needs to
deliver greater output with reduced inputs. In addition to
traditional and marker assisted breeding programs there is an
increased need for the identification and application of novel
genes which can broadly impact crop yield as well as reduce the
impact of environmental stress conditions such as drought, frost,
heat and salinity and require fewer chemical inputs such as
fertilizer, herbicides, pesticides and fungicides. For example, the
2010 worldwide biofuel production (mainly supplied by bioethanol
derived from plant carbohydrate sources, such as starch, sugar from
maize, sugarcane and biodiesel from plant oil (from palm and
soybean)) reached 28 billion gallons of output providing roughly
2.7% of the world's fuels for road transport. One of the keys to
achieving higher yield is to enhance the photosynthetic capacity of
plants such that more carbon dioxide is fixed per plant together
with up-regulating key metabolic pathways leading to increased
levels of storage carbohydrates such as starch and sucrose or
lipids such as fatty acids and triglycerides (oils) in plant
tissues. In the case of biomass crops used for forage or energy
production, increasing the total biomass per plant is also a highly
desirable outcome. In many cases efforts to increase storage
carbohydrates or oil in plants have been focused on genetic
modification using genes encoding individual enzymes in specific
metabolic pathways i.e. "single enzyme" or metabolic pathway
approaches.
Transcription factors (TFs) are considered potential alternatives
to "single enzyme" approaches for the manipulation of plant
metabolism (Grotewold, 2008, Curr. Opin. Biotechnol. 19: 138-144).
They are critical regulators of differential gene expression during
plant growth, development and environmental stress responses.
Transcription factors either directly interact with genes involved
in key biological processes or interact with the regulation of
other TFs that then bind to target genes thus achieving high levels
of specificity and control. The resulting outcome is a multilayered
regulatory network that affects multiple genes and leads to, for
example, fine-tuned changes in the flux of key metabolites through
interconnected or competing metabolic pathways (Ambavaram et al.,
2011, Plant Physiol. 155: 916-931). There is limited information on
transcription factors directly involved in the regulation of
photosynthesis-related genes in plants, improvement of
photosynthetic parameters has been reported in transgenic crop and
model plants overexpressing members of the AP2/EREB, bZIP, NF-X1,
NF-Y(HAP), and MYB families of TFs (Saibo et al., 2009, Ann.
Bot.-London 103: 609-623). Most of these TFs are stress-induced and
confer tolerance to an array of abiotic stress factors, such as
drought, salinity, high or low temperatures, and photoinhibition
(Hussain et al., 2011, Biotechnology Prog. 27: 297-306, see also WO
2005/112608 A2 and U.S. Pat. No. 6,835,540 B2 to Broun). Only a few
TFs, such as Dof1 and MNF from maize are associated with expression
of genes involved in C.sub.4 photosynthesis (Weissmann &
Brutnell, 2012, Curr. Opin. Biotechnol. 23: 298-304; Yanagisawa,
2000, Plant J. 21: 281-288). Increased growth of different
vegetative and/or floral organs resulting in improved biomass
production have been reported in plants overexpressing TFs, such as
ARGOS, AINTEGUMENTA, NAC1, ATAF2, MEGAINTEGUMENTA, and ANGUSTIFOLIA
(Rojas et al., 2010, GM Crops 1: 137-142 and references therein;
see also WO 2011/109661 A1, WO 2010/129501, WO 2009/040665 A2, WO
02/079403 A2 and U.S. Pat. Nos. 7,598,429 B2 to Heard et al. and
7,592,507 B2 to Beekman et al.). Modifications of plant metabolic
pathways by altering the expression of transcription factors
regulating genes in the biosynthesis of lignin (US 2012/0117691 A1
to Wang et al.) and secondary metabolites (U.S. Pat. No. 6,835,540
B2 to Broun) have also been reported.
Thus, a need exists for identification of transcription factors
whose increased or modified expression not only results in
increased levels of the light harvesting pigments used in
photosynthesis and improved photosynthetic capacity of the plants
but which also up-regulate key metabolic pathways resulting in one
or more additional desirable effects selected from the group
comprising: increased levels of starch, glucose or sucrose
(non-structural carbohydrates) in plant tissues; increased levels
of fatty acids; increased production of biomass and/or grain yield;
and enhanced stress tolerance. It is also desirable to be able to
identify suitable variants of such transcription factors in a wide
range of crop species and to be able to engineer these genes in a
wide range of crops including dicots and monocots with C.sub.3 or
C.sub.4 photosynthetic pathways.
Specific crops of interest for practicing this invention include:
switchgrass, Miscathus, Medicago, sweet sorghum, grain sorghum,
sugarcane, energy cane, elephant grass, maize, wheat, barley, oats,
rice, soybean, oil palm, safflower, sesame, flax, cotton,
sunflower, Camelina, Brassica napus, Brassica carinata, Brassica
juncea, pearl millet, foxtail millet, other grain, oilseed,
vegetable, forage, woody and biomass crops.
SUMMARY OF THE INVENTION
This invention is generally in the area of novel genes and methods
for increasing plant crop yield using those novel genes. Described
herein is the use of novel transcription factors that when
overexpressed in a plants of interest affect the regulation of
multiple biological pathways in the crop resulting in, for example,
higher levels of photosynthetic pigments in green tissue, increased
photosynthetic efficiency, increased content of non-structural
carbohydrates (starch, sucrose, glucose) and fatty acids in leaf
tissues, increased biomass yield and improved stress tolerance.
Screening of a number of transcription factor candidates has
resulted in the identification of novel transcription factors that
when expressed from a heterologous promoter in transgenic plants
results in plants having increased expression of these
transcription factors. The increased expression levels can be up to
1.2 fold 1.3 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6
fold, 7 fold, 8 fold, a 9 fold or greater than 10 fold the level of
background expression found in a wild-type plant (e.g.,
non-transgenic plant, test plant or control plant). As a result of
the increased expression of these transcription factors a number of
beneficial traits are achieved including but not limited to:
increased levels of photosynthetic pigments; increased
photosynthetic capacity; increased levels of non-structural
carbohydrates, including starch, sucrose and glucose in plant
tissues; increased levels of fatty acids in plant tissues;
increased biomass growth rate and yield; and improved stress
tolerance in comparison to wild-type plants. Methods for
identifying transcription factors and producing the transgenic
plants are also described herein. The transcription factor genes,
their homologs and/or orthologs and the methods described herein
for increasing their expression or for expressing them in
heterologous hosts can achieve yield improvements in a wide range
of crop plants.
A higher photosynthesis rate in plants transformed with the
transcription factors of the invention and their homologs and/or
orthologs combined with elevated levels of photosynthetic pigments
achieved by the methods described lead to increased accumulation of
products of the central carbon metabolism, such as starch, soluble
sugars and fatty acids as well as improved biomass and grain
production. It is also likely that plants with elevated levels of
expression of these transcription factors will also be useful for
increasing the production of other products produced in plants by
genetic engineering including for example, storage starches. The
overall potential impact of increasing the expression of these
transcription factors in plants is illustrated in FIG. 1. Improved
stress tolerance mediated by the transcription factors of the
invention, produce transgenic plants with better agronomic
performance under abiotic and biotic stress conditions than
non-transformed controls or test plants (also referred to as wild
type). In another related aspect, a quick and reliable method for
testing the stress response of large populations of transgenic and
wild type plants (e.g., crops) is also described. Also described
herein are novel gene sequences, polypeptides encoded by them, gene
constructs and methods for their use to produce transgenic plants,
plant products, crops and seeds.
These transgenic plants, portions of transgenic plants, transgenic
crops and transgenic seeds generated by the introduction of or
increased expression of the functional transcription factors and
their homologs, orthologs and function fragments identified herein
have improved photosynthetic capacity, improved biomass production,
and/or improved grain yield and stress tolerances compared to
wild-type plants.
This invention relates to the identification of transcription
factor genes which when expressed to higher levels than is found in
wild type plants or expressed in heterologous plants results in one
or more desirable traits selected from: higher levels of
photosynthetic pigments; higher photosynthetic activity; higher
levels of starch and/or sucrose and/or glucose; higher yield of
biomass; and improved stress tolerances.
In one aspect of the invention, genes encoding transcription
factors belonging to the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR
(ERF) family (e.g., SEQ ID NOs: 1 and 2) and transcription factors
from the Nuclear-Factor Y (NF-YB) family (e.g., SEQ ID NO: 3) and
their homologues and orthologs from other plant species are
described as well as methods of producing transgenic plants
overexpressing these transcription factors genes in a wide range of
plants to achieve one or more traits selected from: higher levels
of photosynthetic pigments; higher photosynthetic activity; higher
levels of starch and/or sucrose and/or glucose; higher yield, and
improved stress tolerance.
Host plants include but are not limited to food crops, forage
crops, bioenergy and biomass crops, perennial and annual plant
species. Examples of specific crops of interest for practicing this
invention include: switchgrass, Miscathus, Medicago, sweet sorghum,
grain sorghum, sugarcane, energy cane, elephant grass, maize,
wheat, barley, oats, rice, soybean, oil palm, safflower, sesame,
flax, cotton, sunflower, Camelina, Brassica napus, Brassica
carinata, Brassica juncea, pearl millet, foxtail millet, other
grain, oilseed, vegetable, forage, woody and biomass crops.
In a first aspect, a transgenic plant, or a portion of a plant, or
a plant material, or a plant seed, or a plant cell comprising one
or more nucleotide sequences encoding one or more AP2/ERF and/or
NF-YB transcription factors, wherein the AP2/ERF transcription
factor is encoded by the nucleotide sequence of SEQ ID NOs: 1 or 2
and the NF-YB transcription factor is encoded by the nucleotide
sequence of SEQ ID NO: 3 and the increased expression of one or
more transcription factors is increased resulting in one or more
traits selected from: higher levels of photosynthetic pigments;
higher photosynthetic activity; higher levels of starch and/or
sucrose and/or glucose; higher yield; and improved stress tolerance
in the transgenic plant, portion of a plant, plant material, plant
seed, or plant cell is described. The increased expression of the
transcription factors can be measured in a number of ways including
a fold increase over the wild type plant such as 1.5 fold, 2 fold,
3 fold, 4 fold, 5 fold 6 fold 7 fold 8 fold greater than 9 fold
higher than the expression of the same gene in a wild type plant.
In some cases the increased expression results from the expression
of the transcription factor gene through genetic manipulation to
express the transcription factor in a heterologous plant host. An
example of this particular embodiment would be expressing one of
the genes, including homolog or orthologs, isolated from
switchgrass in a plant selected from Miscathus, Medicago, sweet
sorghum, grain sorghum, sugarcane, energy cane, elephant grass,
maize, wheat, barley, oats, rice, soybean, oil palm, safflower,
sesame, flax, cotton, sunflower, Camelina, Brassica napus, Brassica
carinata, Brassica juncea, pearl millet, foxtail millet, other
grain, oilseed, vegetable, forage, woody and biomass crops.
In a first embodiment of the first aspect, the expression of the
one or more transcription factors increases the level of
photosynthetic pigments including chlorophyll and/or carotenoids.
The improvement is compared to a non-transgenic plant and such
improvement can be measured in a variety of ways, including a fold
increase or percent increase, such as 10%, 20%, 50% or 75%.
In a second embodiment of the first aspect, as compared to the wild
type plant, the increased expression of the one or more
transcription factors improves the rate of photosynthesis in the
plant. The improvement is compared to a non-transgenic plant and
such improvement can be measured in a variety of ways, including a
fold increase or percent increase, such as 10%, 20%, 30%, 40%, 50%
or higher.
In a third embodiment of the first aspect, as compared to the wild
type plant, the increased expression of one or more transcription
results in increased levels of starch and/or sucrose and/or glucose
in the plant tissue. The increase in levels of starch and/or
sucrose and/or glucose in the plant tissue alone or in combination
can be measured as a % of dry weight of the plant tissue analyzed
for example 2%, 3%, 4%, 5%, 10%, 15%, 20% of the dry weight of the
plant tissue.
In a fourth embodiment as compared to the wild type plant, the
expression of the one or more transcription factors results in
plants with higher biomass yields. The improvement is compared to a
non-transgenic plant and such improvement can be measured in a
variety of ways, percent increase such as 10%, 20%, 50% or greater
than 50% increase in the dry weight of the plant as compared to a
wild type plant.
In a fifth embodiment as compared to the wild type plant, the
expression of one or more transcription factors improves tolerance
to one or more abiotic stress factors selected from excess or
deficiency of water and/or light, high or low temperature, and high
salinity. The improvement is compared to a non-transgenic plant and
such improvement can be measured in a variety of ways, including a
fold increase or percent increase, such as 10%, 20%, 50% or
75%.
In a second embodiment of the first aspect or of the first
embodiment, the transcription factor is encoded by an ortholog,
homolog, or functional fragment of SEQ ID NOs: 1, 2, or 3. In a
third embodiment of the first aspect or other embodiment, a
promoter is operably linked to one or more nucleotide sequence of
SEQ ID NOs: 1, 2, or 3 in a plant transformation vector.
In a third embodiment of the first aspect or other embodiment, the
plant has increased starch content, soluble sugar content, grain
yield, plant size, organ size, leaf size, and/or stem size when
compared to a non-transgenic plant.
In a fourth embodiment of the first aspect or other embodiment, the
expression of one or more transcription factors increases the
production of food crops, feed crops, or crops used in the
production of fuels or industrial products, when compared to a
non-transgenic plant.
In a second aspect, an isolated nucleotide sequence comprising a
nucleic acid sequence encoding an AP2/ERF or an NF-YB transcription
factor; wherein the transcription factor is functional in a plant,
selected from the group consisting of SEQ ID NOs: 1, 2, and 3; and
expression of the transcription factor result in higher levels of
starch and/or sucrose and/or glucose in the plant.
In a first embodiment of the second aspect, the expression
resulting in higher levels of one or more of starch, sucrose and
glucose and higher biomass, or higher levels of one or more of
starch, sucrose and glucose with no significant increase in
biomass.
In a second embodiment of the second aspect or of the first
embodiment of the second aspect, the nucleic acid sequence further
comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
sequence identity to SEQ ID NOs: 1, 2, or 3.
In a third embodiment of the second aspect or the embodiments, the
plant further comprises a temporal promoter for expression of all
transcription factors such that the gene is overexpressed one the
plant is fully grown and the accumulation of storage materials in
the seed is initiated. Methods of screening for plants with this
outcome are also contemplated. Alternatively, other select
promoters for desirable expression of the transcription factors are
contemplated.
In a fourth embodiment of the second aspect or of the embodiments,
the expression of the transcription factor increases photosynthetic
activity, carbon flow and/or total content of photosynthetic
pigments when compared to a non-transgenic plant.
In a fifth embodiment of the second aspect or of any of the other
embodiments, the nucleic acid sequence encoding a polypeptide of
SEQ ID NOs: 4, 5, or 6.
In a third aspect, a transcription factor, comprising an AP2/ERF or
a NF-YB transcription factor polypeptide selected from SEQ ID NOs:
4, 5, and 6; wherein the transcription factor is functional in a
plant and the expression of the transcription factor increases a
carbon flow in the transgenic plant is described.
In a first embodiment of the third aspect, the transcriptional
factor is functional in a C.sub.3 or C.sub.4 dicotyledonous plant,
a C.sub.3 or C.sub.4 monocotyledonous plant, In a second embodiment
of the third aspect or of any of the other embodiments, the
polypeptide sequence further comprises at least 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 4, 5,
or 6.
In a third embodiment of the third aspect or of any of the other
embodiments, the increased carbon flow is due to increased biomass
yield, or increased starch, glucose or sucrose in plant tissues
when compared to a non-transgenic plant.
In a fourth embodiment of the third aspect or of any of the other
embodiments, expression the transcription factor increases
photosynthetic activity, carbon flow and/or total content of
photosynthetic pigments when compared to a non-transgenic
plant.
In a fourth aspect, a biobased transgenic plant product obtained
from the transgenic plant of the first aspect and any of the
embodiment described having a 100% biobased carbon flow is
described. In certain embodiments of this fourth aspect, the
product is an article having a biobased content of at least 50%, at
least 60%, at least 70%, at least 75%, at least 80%, at least 85%,
90% or 95%.
In a fifth aspect, a method of producing a transgenic plant,
comprising coexpressing one or more AP2/ERF and a NF-YB
transcription factor, wherein the AP2/ERF transcription factor is
encoded by the nucleotide sequence of SEQ ID NOs: 1 or 2 and the
NF-YB transcription factor is encoded by the nucleotide sequence of
SEQ ID NO: 3 is described.
In a sixth aspect, a method for testing the response of plants to
different abiotic stress factors in tissue culture for
identification of plants with increased tolerance to the stress
factors, comprising comparing a test plant with the transgenic
plant of claim 1 under one or more conditions that cause stress
including adverse changes in water, light, temperature, and
salinity is described.
In a seventh aspect methods for transformation comprising
incorporating into the genome of a plant with one or more vectors
comprising the nucleotide sequences described herein are
described.
In an eighth aspect or of any of the embodiments of the first
aspect, the transgenic plant of the first aspect has an increased
photochemical quantum yield than the yield of a non-transgenic
plant.
In a ninth aspect or of any of the embodiments of the first aspect,
the transgenic plant of the first aspect has a starch content
(e.g., yield) increased by at least 2 fold greater than the
corresponding starch content of a non-transgenic plant.
In a tenth aspect or of any of the embodiments of the first aspect,
the transgenic plant of the first aspect has a starch content of at
least 2 fold greater to about 4.3 greater than the content of a
non-transgenic plant.
In an eleventh aspect or of any of the embodiments of the first
aspect, the transgenic plant of the first aspect has a chlorophyll
content that is greater than the content of a non-transgenic plant
or has a chlorophyll content that is at least 1.1 greater to about
2.5-fold greater than the content of a non-transgenic plant.
In a twelfth aspect or of any of the embodiments of the first
aspect, the transgenic plant of the first aspect has a sucrose
content that is higher than the content of a non-transgenic plant
or a sucrose content that is at least two fold greater to about 4.3
fold greater than the content of a non-transgenic plant.
In a thirteenth aspect or of any of the embodiments of the first
aspect, the transgenic plant of the first aspect has an electron
transport rate above the rate of a non-transgenic plant.
In a further embodiment of any of the aspects, the plant is
selected from switchgrass, Miscathus, Medicago, sweet sorghum,
grain sorghum, sugarcane, energy cane, elephant grass, maize,
wheat, barley, oats, rice, soybean, oil palm, safflower, sesame,
flax, cotton, sunflower, Camelina, Brassica napus, Brassica
carinata, Brassica juncea, pearl millet, foxtail millet, other
grain, oilseed, vegetable, forage, industrial, woody and biomass
crops.
In a further embodiment, transgenic plants of the previous
embodiments can be screened to identify plants where the overall
biomass yield is similar to the wild type plant but the levels of
one or more traits selected from: increased concentration of
photosynthetic pigments; increased photosynthesis efficiency;
increased levels of starch and/or sucrose and/or glucose; increased
levels of fatty acids and increased stress tolerance higher than
the levels in the wild-type plants. For example a transgenic plant
with a biomass yield similar to a wild type plant but with a
cumulative level of starch plus glucose plus sucrose 1.5 fold, 2
fold, 5 fold, 10 fold or more higher can be identified.
In a further embodiment, a screening method for identifying
specific genes or combinations of genes which can be used to
achieve some of the individual trait improvements is described
herein.
In certain embodiments, methods related to upregulation of the
central carbon metabolism by PvSTR1, PvSTIF1 and PvBMY1 leading to
increased photosynthetic pigments and activity and elevated levels
of starch, soluble sugars and fatty acids as well as improved
stress tolerance and productivity of plants and plant products are
described. These methods include the incorporation of one or more
of the transcription factors described by SEQ ID NOs: 1, 2 and 3
and homologs, orthologs and functional fragments thereof. For
example, the transgenic plant can comprise SEQ ID NO: 1, SEQ ID NO:
2 or SEQ ID NO: 3, or a homolog, ortholog or functional fragment
thereof or any combination of two or more of SEQ ID NO: 1, SEQ ID
NO: 2 and SEQ ID NO: 3, including their homologs, orthologs or
functional fragments thereof (e.g., SEQ ID NO: 1 and SEQ ID NO: 2;
SEQ ID NO: 1 and SEQ ID NO: 3; homolog of SEQ ID NO: 1 and SEQ ID
NO: 2; homolog of SEQ ID NO: 1 and a homolog of SEQ ID NO: 2;
etc.).
In a fourteenth aspect of the invention, a transgenic plant, or a
portion of a plant, or a plant material, or a plant seed, or a
plant cell comprising one or more nucleotide sequences encoding a
family of AP2/ERF or NF-YB transcription factor, wherein the
AP2/ERF transcription factor is encoded by the nucleotide sequence
of SEQ ID NOs: 1 or 2 and the NF-YB transcription factor is encoded
by the nucleotide sequence of SEQ ID NO: 3; wherein the expression
of the one or more transcription factors increases carbon flow in
the transgenic plant, portion of a plant, plant material, plant
seed, or plant cell is described. In a first embodiment of the
fourteenth aspect, the expression of the one or more transcription
factors improves tolerance to one or more abiotic stress factors
selected from excess or deficiency of water and/or light, from high
or low temperature, and high salinity. In a second embodiment of
the fourteenth aspect or of the first embodiment of the aspect, the
transcription factor is encoded by an ortholog, homolog, or
functional fragment encoded by SEQ ID NOs: 1, 2, or 3. In a third
embodiment of the fourteenth aspect or of any of the embodiments of
the aspect, the transgenic plant, portion of a plant or plant
material, plant seed or plant cell, further comprises a vector
containing a promoter operably linked to one or more nucleotide
sequence of SEQ ID NOs: 1, 2, or 3. In a fourth embodiment of the
fourteenth aspect or of any of the embodiments of the aspect the
plant is selected from a crop plant, a model plant, a
monocotyledonous plant, a dicotyledonous plant, a plant with C3
photosynthesis, a plant with C4 photosynthesis, an annual plant, a
perennial plant, a switchgrass plant, a maize plant, or a sugarcane
plant. In a fifth embodiment of the fourteenth aspect or of any of
the embodiments of the aspect the annual or perennial plant is a
bioenergy or biomass plant. In a sixth embodiment of the fourteenth
aspect or of any of the embodiments of the aspect expression of one
or more transcription factors increases photosynthetic activity,
carbon flow and/or total content of photosynthetic pigments. In a
seventh embodiment of the fourteenth aspect or of any of the
embodiments of the aspect the increased carbon flow results in
increased biomass yield when compared to a non-transgenic plant. In
an eighth embodiment of the fourteenth aspect or of any of the
embodiments of the aspect, wherein the plant has an increase of one
or more of the following: starch content, soluble sugars content,
grain yield, plant size, organ size, leaf size, and/or stem size
when compared to a non-transgenic plant. In a ninth embodiment of
the fourteenth aspect or of any of the embodiments of the aspect
the expression of one or more transcription factors leads to
increases in the production of food crops, feed crops, or crops for
the production of fuels or industrial products, when compared to a
non-transgenic plant.
In a fifteenth aspect of the invention, an isolated nucleotide
sequence comprising a nucleic acid sequence encoding an AP2/ERF or
an NF-YB transcription factor; wherein the transcription factor
selected from the group consisting of SEQ ID NOs: 1, 2, and 3 is
functional in a plant; and expression of the transcription factor
increases carbon flow in the transgenic plant is described. In a
first embodiment of the fifteenth aspect, the plant is selected
from the group consisting of a C3 or C4 dicotyledonous plant, a C3
or C4 monocotyledonous plant, grass, a switchgrass plant, a maize
plant, or a sugarcane plant. In a second embodiment of the
fifteenth aspect or of any of the embodiments of the aspect, the
nucleic acid sequence further comprises at least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 1,
2, or 3. In a third embodiment of the fifteenth aspect or of any of
the embodiments of the aspect, the increased biomass yield is due
to increased carbon flow when compared to a non-transgenic plant.
In a fourth embodiment of the fifteenth aspect or of any of the
embodiments of the aspect, expression of the transcription factor
increases photosynthetic activity, carbon flow and/or total content
of photosynthetic pigments when compared to a non-transgenic plant.
In a fifth embodiment of the fifteenth aspect or of any of the
embodiments of the aspect, the nucleic acid sequence encodes a
polypeptide of SEQ ID NOs: 4, 5, or 6. In a sixth embodiment of the
fifteenth aspect or of any of the embodiments of the aspect, the
increased carbon flow increases the starch, sucrose and glucose
levels in a transgenic plant without the same corresponding
increase in biomass yield.
In a sixteenth aspect, a transcription factor, comprising an
AP2/ERF or a NF-YB transcription factor polypeptide selected from
SEQ ID NOs: 4, 5, and 6; wherein the transcription factor is
functional in a plant and the expression of the transcription
factor increases a carbon flow in the transgenic plant is
described. In a first embodiment of the sixteenth aspect, the plant
is selected from the group consisting of a C3 or C4 dicotyledonous
plant, a C3 or C4 monocotyledonous plant, grass, or a switchgrass
plant, a maize plant, or a sugarcane plant. In a second embodiment
of the sixteenth aspect or of the first embodiment of the aspect,
the polypeptide sequence further comprises at least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 4,
5, or 6. In a third embodiment of the sixteenth aspect or of the
first or second embodiment of the aspect, the increased biomass
yield is due to increased carbon flow when compared to a
non-transgenic plant. In a fourth embodiment of the sixteenth
aspect or of the first, second or third embodiment of the aspect,
expression of the transcription factor increases photosynthetic
activity, carbon flow and/or total content of photosynthetic
pigments when compared to a non-transgenic plant.
In a seventeenth aspect, a method for manufacturing a transgenic
seed for producing a crop of transgenic plants with an enhanced
trait resulting from the expression of one or more transcription
factors or homologs, orthologs or functional fragments thereof,
encoded by the nucleotide sequence of SEQ ID NOs: 1, 2 or 3,
comprising: a) screening a population of plants transformed with
transcription factor(s) for the enhanced trait; b) selecting from
the population one or more plants that exhibit the trait; and c)
collecting seed from the selected plant is described. In a first
embodiment of the seventeenth aspect, the seed is maize seed or
sorghum seed and the enhanced trait is seed carbon content.
In an eighteen aspect, a method of producing a transgenic plant,
comprising coexpressing one or more AP2/ERF and NF-YB transcription
factors in a plant, wherein the AP2/ERF transcription factor is
encoded by the nucleotide sequence of SEQ ID NOs: 1 or 2 and the
NF-YB transcription factor is encoded by the nucleotide sequence of
SEQ ID NO: 3 is described.
In a nineteenth aspect, a method for testing the response of a
plant to different stress factors in tissue culture for
identification of plants with increased tolerance to the stress
factors, comprising comparing a test plant with the transgenic
plant of the fourteen aspect under one or more conditions that
cause stress including changes in water, light, temperature, and
salinity is described. In an embodiment of the seventeenth,
eighteen or nineteen aspect, further comprising introducing into a
plant one or more vectors comprising the nucleotide sequences of
the invention.
In any of the aspects or embodiments described above, the
photochemical quantum yield of the plant is at least 2-fold greater
than the yield of a corresponding non-transgenic plant. In any of
the aspects or embodiments described above, the plant has a starch
yield increased by at least 2-fold the content of a corresponding
non-transgenic plant. In any of the aspects or embodiments
described above, the plant has a starch yield increased by at least
2-fold to about a 4.5-fold content of a corresponding
non-transgenic plant. In any of the aspects or embodiments
described above, the plant has a chlorophyll content that is 1.5
times greater than the content of a corresponding non-transgenic
plant. In any of the aspects or embodiments described above, the
plant has a chlorophyll content that is at least 1.5 fold greater
to about 2.5 fold greater than the content of a corresponding
non-transgenic plant. In any of the aspects or embodiments
described above, the plant has a sucrose content that is at least
1.5 fold greater than the content of a corresponding non-transgenic
plant. In any of the aspects or embodiments described above, the
plant has a sucrose content that is at least two fold greater to
about 4.3 fold greater than the content of a corresponding
non-transgenic plant. In any of the aspects or embodiments
described above, the plant has a plant grown rate increased by at
least 10% above the rate of a corresponding non-transgenic plant.
In any of the aspects or embodiments described above, the plant is
switchgrass, maize, or sugar cane.
In a twentieth aspect, a method for enhancing a trait in a
transgenic plant relative to a control non-transgenic plant,
comprising: (a) increasing expression of at least one nucleic acid
sequence encoding a transcription factor from AP2/ERF and NF-YB
families, selected from the nucleic acid sequence of SEQ ID NO: 1,
SEQ ID NO: 2, and SEQ ID NO: 3, or an ortholog, homolog or
functional fragment thereof; and (b) selecting for a transgenic
plant having an enhanced trait relative to a control plant is
described. In a first embodiment of the twentieth aspect, the trait
is selected from one or more of the following: carbon flow, primary
metabolites, tolerance to one or more abiotic stress factors, and
one or more photosynthetic pigments.
In a twenty-first aspect, a transgenic plant having a trait
modification relative to a corresponding non-transgenic plant,
comprising one or more nucleotide sequences encoding a AP2/ERF or
NF-YB transcription factors, wherein the AP2/ERF transcription
factor is encoded by the nucleotide sequence of SEQ ID NOs: 1 or 2
and the NF-YB transcription factor is encoded by the nucleotide
sequence of SEQ ID NO: 3 or a ortholog, homolog, or functional
fragment thereof, wherein the trait modification is selected from
one or more of the following: carbon flow, levels of photosynthetic
pigments; photosynthetic capacity; levels of starch, sucrose and
glucose in plant tissues, levels of fatty acids in plant tissues;
biomass growth rate and yield; and stress tolerance is described.
In a first embodiment of the twenty-first aspect, the trait
modification is a greater than 3 fold yield of starch or soluble
sugars and the increase in biomass production is less than 1.5
fold.
In a twenty-second aspect, a transgenic maize plant having an
increased non-structural carbohydrate content comprising, a)
introducing into a plant cell one or more nucleotides encoding
AP2/ERF and/or NF-YB transcription factor, wherein the AP2/ERF
transcription factor is encoded by the nucleotide sequence of SEQ
ID NOs: 1 or 2 and the NF-YB transcription factor is encoded by the
nucleotide sequence of SEQ ID NO: 3 or a ortholog, homolog, or
functional fragment thereof, and b) producing a transgenic plant
from the plant cell having an increased non-structural carbohydrate
content compared to a corresponding non-transgenic plant is
described. In a first embodiment of the aspect a seed or plant
tissue is obtained by the transgenic maize or sorghum plant.
In a twenty-third aspect, a method of identifying a drought and
salinity resistant transgenic plant having one or more nucleotides
encoding an AP2/ERF and/or NF-YB transcription factor, wherein the
AP2/ERF transcription factor is encoded by the nucleotide sequence
of SEQ ID NOs: 1 or 2 and the NF-YB transcription factor is encoded
by the nucleotide sequence of SEQ ID NO: 3 or a ortholog, homolog,
or functional fragment thereof comprising, (a) growing a population
of transgenic and wild-type plants under conditions of drought and
salinity stress; (b) selecting a transgenic plant that exhibits
tolerance to drought and salinity, thereby identifying a transgenic
plant that comprises a genotype associated with tolerance to
drought and salinity is described.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing will be apparent from the following more particular
description of example embodiments of the invention, as illustrated
in the accompanying drawings in which like reference characters
refer to the same parts throughout the different views. The
drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
FIG. 1 graphically illustrates the transcriptional regulatory
network model of the switchgrass transcription factors PvSTR1,
PvSTIF1 and PvBMY1 and their association to improved plant
productivity and stress tolerance. The thick arrows illustrate the
observed increased carbon flow directly regulated by the
transcription factors, whereas the small arrows indicate the
interactions with downstream TFs for regulation of key genes in
major metabolic pathways.
FIG. 2 illustrates the tissue specific expression pattern of the
transcription factor genes PvSTR1, PvSTIF1 and PvBMY1 in wild type
switchgrass analyzed by RT-PCR. Total RNA was isolated from roots
(R), young leaves (YL), culms (C), mature leaves (ML), leaf sheaths
(LS), and/or panicles (P) of wild-type plants and subjected to
reverse transcription and PCR using One Step RT-PCR Kit (Qiagen)
and primers specific for the coding regions of the TF genes.
FIG. 3 demonstrates the presence of genes homologous to the
transcription factor genes PvSTR1, PvSTIF1 and PvBMY1 in the
switchgrass genome as detected by Southern blot hybridization.
Genomic DNA isolation, digestion with EcoRI and hybridization with
probes specific for the coding regions of the transcription factor
genes was performed as described previously (Somleva et al., 2008,
Plant Biotechnol J, 6: 663-678). 16 and 56, Alamo genotypes (our
designation).
FIG. 4A-C shows the multiple sequence alignment for the conserved
domains of PvSTR1 (FIG. 4A), PvSTIF1 (FIG. 4B) and PvBMY1 (FIG. 4C)
in switchgrass (Panicum virgatum L.) and other plant species. The
alignments of the DNA-binding domain sequences (AP2/ERF for STIF1
and STR1 and NFYB-HAP3 for BMY1) obtained using Clustal W program
(Thompson et al., 1994, Nucleic Acids Res. 11: 4673-4680) are shown
in the boxes.
FIG. 5A-C illustrates the possible phylogenetic relationships among
the higher plant taxa, including monocotyledonous and
dicotyledonous species, based on the conservative domains of PvSTR1
(A), PvSTIF1 (B) and PvBMY1 (C).
FIG. 6A-C shows the vectors pMBXS809 (A), pMBXS810 (B) and pMBXS855
(C) harboring the TF genes and the marker gene bar.
FIG. 7A-C depicts the vectors pMBXS881 (A), pMBXS882 (B) and
pMBXS883 (C) harboring the TF genes and the marker gene hptII.
FIG. 8 shows results from qRT-PCR (quantitative reverse
transcription polymerase chain reaction or real-time RT-PCR)
analysis of the overexpression of the transcription factor genes
PvSTR1 (A), PvSTIF1 (B) and PvBMY1 (C) in transgenic switchgrass
plants prior to transfer to soil. .beta.-actin amplification was
used for transcript normalization. WT1, plants regenerated from
non-transformed mature caryopsis-derived callus cultures from
genotype 16; WT2, plants regenerated from non-transformed immature
inflorescence-derived cultures from genotype 56; 1-5, transgenic
lines representing independent transformation events. Data
presented as mean values.+-.SE (n=3).
FIG. 9 shows Western blots of total proteins from transgenic and
wild-type switchgrass plants. A Protein extracts (6 .mu.g per lane)
from PvSTR1 and PvSTIF1 lines incubated with antibodies against the
proteins of the light harvesting centers of photosystem I (LhcA3)
and photosystem II (LhcB5). .beta.-actin was used as a loading
control. B Total protein extracts (6 .mu.g per lane) from PvBMY1
lines incubated with an antibody against phosphoenolpyruvate
carboxylase (PEPC). .beta.-actin was used as a loading control.
Protein isolation and membrane blotting were performed as described
previously (Somleva et al., 2008, Plant Biotechnol J, 6: 663-678).
Commercially available antibodies (Agrisera) were used for protein
detection. An ultra-sensitive chemiluminescent substrate system
(Thermo Scientific) was used for signal development. Lanes: WT--a
control, wild-type plant; 1 to 4--transgenic switchgrass plants
representing different TF lines.
FIG. 10 illustrates the effect of high salinity stress on relative
water content (A), the abundance of the chloroplastic Cu--Zn
superoxide dismutase (SOD) protein (B) and levels of photosynthetic
pigments (C) in switchgrass plants overexpressing the PvSTR1 and
PvSTIF1 genes. Bars represent mean.+-.SD values (n=3).
FIG. 11 illustrates the large number of switchgrass genes,
including transcription factors whose expression is impacted by
over-expression of PvSTR1, PvSTIF1 and PvBMY1. The data is
presented as the total number of regulated orthologs (A) as well as
the numbers of up-regulated (B) and down-regulated (C) genes common
for the three TFs.
FIG. 12 presents the gene ontology analysis of differentially
expressed genes regulated by PvSTR1 (A), PvSTIF1 (B) and PvBMY1 (C)
transcription factors. Descriptions of biological functions were
assigned on the basis of information retrieved from the world wide
web at: bioinfo.cau.edu.cn/agriGO/index.php (P-value calculated by
Fisher exact test). Genes that showed more than 2-fold
up-regulation and the top enriched pathways are considered for the
graphs.
DETAILED DESCRIPTION OF THE INVENTION
A description of example embodiments of the invention follows.
I. Definitions
Unless otherwise indicated, the disclosure encompasses all
conventional techniques of plant transformation, plant breeding,
microbiology, cell biology and recombinant DNA, which are within
the skill of the art. See, e.g., Sambrook and Russell, Molecular
Cloning: A Laboratory Manual, 3rd edition, 2001; Current Protocols
in Molecular Biology, F. M. Ausubel et al. eds., 1987; Plant
Breeding: Principles and Prospects, M. D. Hayward et al., 1993;
Current Protocols in Protein Science, Coligan et al., eds., 1995,
(John Wiley & Sons, Inc.); the series Methods in Enzymology
(Academic Press, Inc.): PCR 2: A Practical Approach, M. J.
MacPherson, B. D. Hames and G. R. Taylor eds., 1995.
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Lewin, Genes VII, 2001 (Oxford University
Press), The Encyclopedia of Molecular Biology, Kendrew et al.,
eds., 1999 (Wiley-Interscience) and Molecular Biology and
Biotechnology, a Comprehensive Desk Reference, Robert A. Meyers,
ed., 1995 (VCH Publishers, Inc), Current Protocols In Molecular
Biology, F. M. Ausubel et al., eds., 1987 (Green Publishing),
Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd
edition, 2001.
A number of terms used herein are defined and clarified in the
following section.
As used herein, a "vector" is a replicon, such as a plasmid, phage,
or cosmid, into which another DNA segment may be inserted so as to
bring about the replication of the inserted segment. The vectors
described herein can be expression vectors.
As used herein, an "expression vector" is a vector that includes
one or more expression control sequences.
As used herein, an "expression control sequence" is a DNA sequence
that controls and regulates the transcription and/or translation of
another DNA sequence.
As used herein, "operably linked" means incorporated into a genetic
construct so that expression control sequences effectively control
expression of a coding sequence of interest.
As used herein, "transformed" and "transfected" encompass the
introduction of a nucleic acid (e.g., a vector) into a cell by a
number of techniques known in the art.
"Plasmids" are designated by a lower case "p" preceded and/or
followed by capital letters and/or numbers.
The term "plant" is used in its broadest sense. It includes, but is
not limited to, any species of woody, ornamental or decorative,
crop or cereal, fruit or vegetable plant, and photosynthetic green
algae (e.g., Chlamydomonas reinhardtii). It also refers to a
plurality of plant cells that is largely differentiated into a
structure that is present at any stage of a plant's development.
Such structures include, but are not limited to, a fruit, shoot,
stem, leaf, flower petal, etc.
The term "plant tissue" includes differentiated and
undifferentiated tissues of plants including those present in
roots, shoots, leaves, inflorescences, anthers, pollen, ovaries,
seeds and tumors, as well as cells in culture (e.g., single cells,
protoplasts, embryos, callus, etc.). Plant tissue may be in planta,
in organ culture, tissue culture, or cell culture.
The term "plant part" as used herein refers to a plant structure, a
plant organ, or a plant tissue.
A "non-naturally occurring plant" refers to a plant that does not
occur in nature without human intervention. Non-naturally occurring
plants include transgenic plants, plants created through genetic
engineering and plants produced by non-transgenic means such as
traditional or market assisted plant breeding.
The term "plant cell" refers to a structural and physiological unit
of a plant, comprising a protoplast and a cell wall. The plant cell
may be in the form of an isolated single cell or a cultured cell,
or as a part of a higher organized unit such as, for example, a
plant tissue, a plant organ, or a whole plant.
The term "plant cell culture" refers to cultures of plant units
such as, for example, protoplasts, cells and cell clusters in a
liquid medium or on a solid medium, cells in plant tissues and
organs, microspores and pollen, pollen tubes, anthers, ovules,
embryo sacs, zygotes and embryos at various stages of
development.
The term "plant material" refers to leaves, stems, roots,
inflorescences and flowers or flower parts, fruits, pollen,
anthers, egg cells, zygotes, seeds, cuttings, cell or tissue
cultures, or any other part or product of a plant.
A "plant organ" refers to a distinct and visibly structured and
differentiated part of a plant, such as a root, stem, leaf, flower
bud, inflorescence, spikelet, floret, seed or embryo.
The term "non-transgenic plant" refers to a plant that has not been
genetically engineered with heterologous nucleic acids. These
non-transgenic plants can be the test or control plant when
comparisons are made, including wild-type plants.
A "corresponding non-transgenic plant" refers to the plant prior to
the introduction of heterologous nucleic acids. This plant can be
the test plant or control plant, including wild type plants.
A "trait` refers to morphological, physiological, biochemical and
physical characteristics or other distinguishing feature of a plant
or a plant part or a cell or plant material.
The term "trait modification" refers to a detectable change in a
characteristic of a plant or a plant part or a plant cell induced
by the expression of a polynucleotide or a polypeptide of the
invention compared to a plant not expressing them, such as a wild
type plant. Some trait modifications can be evaluated
quantitatively, such as content of different metabolites, proteins,
pigments, lignin, vitamins, starch, sucrose, glucose, fatty acids
and other storage compounds, seed size and number, organ size and
weight, total plant biomass and yield of genetically engineered
products.
Trait modifications of further interest include those to seed (such
as embryo or endosperm), fruit, root, flower, leaf, stem, shoot,
seedling or the like, including: enhanced tolerance to
environmental conditions including freezing, chilling, heat,
drought, water saturation, radiation and ozone; improved growth
under poor photoconditions (e.g., low light and/or short day
length), or changes in expression levels of genes of interest.
Other phenotype that can be modified relate to the production of
plant metabolites, such as variations in the production of
photosynthetic pigments, enhanced or compositionally altered
protein or oil production (especially in seeds), or modified sugar
(insoluble or soluble) and/or starch composition. Physical plant
characteristics that can be modified include cell development (such
as the number of trichomes), fruit and seed size and number, yields
and size of plant parts such as stems, leaves and roots, the
stability of the seeds during storage, characteristics of the seed
pod (e.g., susceptibility to shattering), root hair length and
quantity, internode distances, or the quality of seed coat. Plant
growth characteristics that can be modified include growth rate,
germination rate of seeds, vigor of plants and seedlings, leaf and
flower senescence, male sterility, apomixis, flowering time, flower
abscission, rate of nitrogen uptake, biomass or transpiration
characteristics, as well as plant architecture characteristics such
as apical dominance, branching patterns, number of organs, organ
identity, organ shape or size.
As used herein "abiotic stress" includes but is not limited to
stress caused by any one of the following: drought, salinity,
extremes or atypical temperature, chemical toxicity and oxidative
variation. The ability to improve plant tolerance to abiotic stress
would be of great economic advantage to farmers worldwide and would
allow for the cultivation of crops during adverse conditions and in
territories where cultivation of crops may not otherwise be
possible.
Methods and Transgenic Plants, Plant Tissue, Seed and Plant Cell of
the Invention
Described herein are methods of producing a transgenic plant, plant
tissue, seed, or plant cell, wherein said plant, plant tissue, seed
or plant cell comprises incorporated in the genome of said plant,
plant tissue, seed, or plant cell: a polynucleotide encoding a
plant transcription factor together with sequences to enable its
increased expression or regulatory sequences inserted to increase
the expression of a heterologous plant transcription factor.
It was found that incorporation of transcription factors encoded by
the nucleotides SEQ ID NOs: 1, 2, and 3 modified expression of
certain genes in a transgenic plant and increased the carbon flow
of the transgenic plant without the corresponding increase in
biomass. For example, increases in the levels of non-structural
carbohydrates such as starch, sucrose and glucose levels in a
transgenic plant are found to be greater than 2 fold increase but
without an increase in the biomass or an insignificant increase in
the biomass compared to the increases in the non-structural
carbohydrates.
II. Transcriptional Regulation of Gene Expression in Plants
Transcription factors (TFs) are known to be involved in various
biological processes, acting as activators or repressors of other
genes or gene families, suggesting the function of various
transcriptional regulatory mechanisms in regulating downstream
signal transduction pathways. The regulatory logic that drives any
plant response is governed by the combination of signaling
regulators, TFs, their binding site in the regulatory regions of
target genes (cis-regulatory elements; CREs) and other regulatory
molecules (e.g., chromatin modifiers and small RNAs), as well as
protein and RNA degradation machinery (Krishnan & Pereira,
2008, Brief Funct. Genomic. Proteomic. 7: 264-74). TFs control the
expression of many target genes through specific binding of the TF
to the corresponding CRE in the promoters of respective target
genes. For example, recent reports suggest that the maize Dof1 and
MNF factors bind to the promoter of PEPC, an enzyme in the C.sub.4
cycle of photosynthesis (reviewed in Weissmann & Brutnell,
2012, Current Opinion Biotech. 23: 298-304). Several TFs are known
to be induced by stress, acting as activators or repressors,
suggesting the function of various transcriptional regulatory
mechanisms in regulating specific biological processes and or
pathways.
Identification and Mapping Regulatory Domains of TFs:
Targeted gene regulation via designed transcription factors has
great potential for precise phenotypic modification and
acceleration of novel crop trait development. Over the past few
years many transcription factors have been shown to contain
regulatory domains, which can increase or decrease their
transcriptional and/or DNA-binding activity. The mechanisms by
which this regulation takes place frequently involve
phosphorylation, dimer formation or interaction with negative or
positive cofactors (Facchinetti et al., 1997, Biochem. J. 324:
729-736). Nevertheless, different organisms have evolved with
diverse temporal and spatial regulation of transcription. In
general, the temporal and spatial regulations are mediated by
different classes of DNA binding transcriptional activator
proteins. Unlike DNA binding domains, the transcription activation
domains (TAD) have less primary amino acid sequence similarity. The
TADs have been classified into acidic, glutamine-rich, proline-rich
and serine/threonine-rich. We have identified putative
transcription activation domains of the transcription factors of
the invention based on the bioinformatics analysis.
Spatio-Temporal Gene Expression Through Novel cis-Regulatory
Elements:
Spatio-temporal gene expression is the activation of genes within
specific tissues of an organism at specific times during
development. Plant promoters have attracted increasing attention
because of their irreplaceable role in modulating the
spatio-temporal expression of genes interacting with transcription
factors (TFs). The control of gene expression is largely determined
by cis-regulatory modules localized in the promoter sequence of
regulated genes and their cognate transcription factors. While
there has been a substantial progress in dissecting and predicting
cis-regulatory activity, our understanding of how information from
multiple enhancer elements converge to regulate a gene's expression
remains elusive. Constitutive promoters are widely used to
functionally characterize plant genes in transgenic plants but
their lack of specificity and poor control over protein expression
can be a major disadvantage. On the other hand, promoters that
provide precise regulation of temporal or spatial transgene
expression facilitate such studies by targeting overexpression or
knockdown of target genes to specific tissues and/or at particular
developmental stages. Promoter-based transgenic technologies have
already been applied to a great effect in wheat, where a
heat-inducible promoter in transgenic plants effectively controlled
the spatio-temporal expression of a transgene (Freeman et al.,
2011, Plant Biotech. J. 9: 788-796). A modular synthetic promoter
for the spatio-temporal control of transgene expression in stomata
has been reported by fusing a guard cell-specific element from the
promoter of the potato phosphoenolpyruvate carboxylase (PEPC) gene
with the ethanol-inducible gene switch AlcR/alcA (Xiong et al.,
2009, J. Exp. Bot. 60: 4129-4136). Recently, a chimeric inducible
system was developed, which combined the cellular specificity of
the AtMYB60 minimal promoter with the positive responsiveness to
dehydration and ABA of the rd29A promoter (Rusconi et al., 2013, J.
Exp. Bot. 64: 3361-3371). Remarkably, the synthetic module
specifically up-regulated gene expression in guard cells of
Arabidopsis, tobacco, and tomato in response to dehydration or ABA.
Likewise, promoter cloning and subsequent manipulation of
spatio-temporal gene expression together with transcription
activation domains from the switchgrass transcription factors
described in the presented invention offers a significant promise
in genetically engineering novel adaptive traits in biomass and
bioenergy crops.
III. Plant Transformation Technologies
The transcription factor genes of this invention can be introduced
into the genome of any plant by any of the methods for nuclear
transformation known in the art. Methods for transformation of a
range of plants useful for practicing the current invention are
described in the examples herein. Any other genes of interest can
be introduced into the genome and/or plastome of any plant by any
of the methods for nuclear and plastid transformation known in the
art. Other genes of interest can include herbicide resistance
genes, pest resistance genes, fungal resistance genes, genes for
enhancing oil yield or genes for novel metabolic pathways enabling
the production of non-plant products to be made by the plant. The
product of any transgene can be targeted to one or more of the
plant cell organelles using any of the targeting sequences and
methods known in the art.
A. Genetic Constructs for Transformation
DNA constructs useful in the methods described herein include
transformation vectors capable of introducing transgenes into
plants. As used herein, "transgenic" refers to an organism in which
a nucleic acid fragment containing a heterologous nucleotide
sequence has been introduced. The transgenes in the transgenic
organism are preferably stable and inheritable. The heterologous
nucleic acid fragment may or may not be integrated into the host
genome.
Several plant transformation vector options are available,
including those described in Gene Transfer to Plants, 1995,
Potrykus et al., eds., Springer-Verlag Berlin Heidelberg New York,
Transgenic Plants: A Production System for Industrial and
Pharmaceutical Proteins, 1996, Owen et al., eds., John Wiley &
Sons Ltd. England, and Methods in Plant Molecular Biology: A
Laboratory Course Manual, 1995, Maliga et al., eds., Cold Spring
Laboratory Press, New York. Plant transformation vectors generally
include one or more coding sequences of interest under the
transcriptional control of 5' and 3' regulatory sequences,
including a promoter, a transcription termination and/or
polyadenylation signal, and a selectable or screenable marker gene.
For the expression of two or more polypeptides from a single
transcript, additional RNA processing signals and ribozyme
sequences can be engineered into the construct (U.S. Pat. No.
5,519,164). This approach has the advantage of locating multiple
transgenes in a single locus, which is advantageous in subsequent
plant breeding efforts.
Engineered minichromosomes can also be used to express one or more
genes in plant cells. Cloned telomeric repeats introduced into
cells may truncate the distal portion of a chromosome by the
formation of a new telomere at the integration site. Using this
method, a vector for gene transfer can be prepared by trimming off
the arms of a natural plant chromosome and adding an insertion site
for large inserts (Yu et al., 2006, Proc. Natl. Acad. Sci. USA 103:
17331-17336; Yu et al., 2007, Proc. Natl. Acad. Sci. USA 104:
8924-8929).
An alternative approach to chromosome engineering in plants
involves in vivo assembly of autonomous plant minichromosomes
(Carlson et al., 2007, PLoS Genet. 3: 1965-74). Plant cells can be
transformed with centromeric sequences and screened for plants that
have assembled autonomous chromosomes de novo. Useful constructs
combine a selectable marker gene with genomic DNA fragments
containing centromeric satellite and retroelement sequences and/or
other repeats.
Another approach useful to the described invention is Engineered
Trait Loci ("ETL") technology (U.S. Pat. No. 6,077,697; US
2006/0143732). This system targets DNA to a heterochromatic region
of plant chromosomes, such as the pericentric heterochromatin, in
the short arm of acrocentric chromosomes. Targeting sequences may
include ribosomal DNA (rDNA) or lambda phage DNA. The pericentric
rDNA region supports stable insertion, low recombination, and high
levels of gene expression. This technology is also useful for
stacking of multiple traits in a plant (US 2006/0246586).
Zinc-finger nucleases (ZFNs) are also useful for practicing the
invention in that they allow double strand DNA cleavage at specific
sites in plant chromosomes such that targeted gene insertion or
deletion can be performed (Shukla et al., 2009, Nature 459:
437-441; Townsend et al., 2009, Nature 459: 442-445). This approach
may be particularly useful for the present invention which can
involve transcription factor genes which are naturally present in
the genome of the plant of interest. In this case the ZFNs can be
used to change the sequences regulating the expression of the TF of
interest to increase the expression or alter the timing of
expression beyond that found in a non-engineered or wild type
plant.
A transgene may be constructed to encode a multifunctional
transcription factor combining different domains of the
transcription factors identified herein as useful for practicing
the claimed invention through gene fusion techniques in which the
coding sequences of different domains of the different genes are
fused with or without linker sequences to obtain a single gene
encoding a single protein with the activities of the individual
genes. Such synthetic fusion gene/TF combinations can be further
optimized using molecular evolution technologies.
B. Tissue Culture-Based Methods for Nuclear Transformation
Transformation protocols as well as protocols for introducing
nucleotide sequences into plants may vary depending on the type of
plant or plant cell, i.e., monocot or dicot, targeted for
transformation.
Suitable methods of introducing nucleotide sequences into plant
cells and subsequent insertion into the plant genome are described
in US 2010/0229256 A1 to Somleva & Ali and US 2012/0060413 to
Somleva et al.
The transformed cells are grown into plants in accordance with
conventional techniques. See, for example, McCormick et al., 1986,
Plant Cell Rep. 5: 81-84. These plants may then be grown, and
either pollinated with the same transformed variety or different
varieties, and the resulting hybrid having constitutive expression
of the desired phenotypic characteristic identified. Two or more
generations may be grown to ensure that constitutive expression of
the desired phenotypic characteristic is stably maintained and
inherited and then seeds harvested to ensure constitutive
expression of the desired phenotypic characteristic has been
achieved.
C. In Planta Transformation Methods
Procedures for in planta transformation can be simple. Tissue
culture manipulations and possible somaclonal variations are
avoided and only a short time is required to obtain transgenic
plants. However, the frequency of transformants in the progeny of
such inoculated plants is relatively low and variable. At present,
there are very few species that can be routinely transformed in the
absence of a tissue culture-based regeneration system. Stable
Arabidopsis transformants can be obtained by several in planta
methods including vacuum infiltration (Clough & Bent, 1998, The
Plant J. 16: 735-743), transformation of germinating seeds
(Feldmann & Marks, 1987, Mol. Gen. Genet. 208: 1-9), floral dip
(Clough and Bent, 1998, Plant J. 16: 735-743), and floral spray
(Chung et al., 2000, Transgenic Res. 9: 471-476). Other plants that
have successfully been transformed by in planta methods include
rapeseed and radish (vacuum infiltration, Ian and Hong, 2001,
Transgenic Res., 10: 363-371; Desfeux et al., 2000, Plant Physiol.
123: 895-904), Medicago truncatula (vacuum infiltration, Trieu et
al., 2000, Plant J. 22: 531-541), camelina (floral dip,
WO/2009/117555 to Nguyen et al.), and wheat (floral dip, Zale et
al., 2009, Plant Cell Rep. 28: 903-913). In planta methods have
also been used for transformation of germ cells in maize (pollen,
Wang et al. 2001, Acta Botanica Sin., 43, 275-279; Zhang et al.,
2005, Euphytica, 144, 11-22; pistils, Chumakov et al. 2006, Russian
J. Genetics, 42, 893-897; Mamontova et al. 2010, Russian J.
Genetics, 46, 501-504) and Sorghum (pollen, Wang et al. 2007,
Biotechnol. Appl. Biochem., 48, 79-83)
D. Transformation of Plants with Genes of Interest
Transgenic plants can be produced using conventional techniques to
express any genes of interest in plants or plant cells (Methods in
Molecular Biology, 2005, vol. 286, Transgenic Plants: Methods and
Protocols, Pena L., ed., Humana Press, Inc. Totowa, N.J.).
Typically, gene transfer, or transformation, is carried out using
explants capable of regeneration to produce complete, fertile
plants. Generally, a DNA or an RNA molecule to be introduced into
the organism is part of a transformation vector. A large number of
such vector systems known in the art may be used, such as plasmids.
The components of the expression system can be modified, e.g., to
increase expression of the introduced nucleic acids. For example,
truncated sequences, nucleotide substitutions or other
modifications may be employed. Expression systems known in the art
may be used to transform virtually any plant cell under suitable
conditions. A transgene comprising a DNA molecule encoding a gene
of interest is preferably stably transformed and integrated into
the genome of the host cells. Transformed cells are preferably
regenerated into whole plants. Detailed description of
transformation techniques are within the knowledge of those skilled
in the art.
1. Genes for Transcription Factors
Crop improvement using transcription factors (TFs) is a promising
approach as they are likely to regulate a wide range of target
genes whose products contribute to plant agronomic performance
under normal and stress conditions. TF-mediated improvement of
stress tolerance has been reported in diverse crop species, both
dicots and monocots (Hussain et al., 2011, Biotechnology Prog. 27:
297-306). The first efforts included overexpression of the AP2/ERF
factors CBF1, DREB1A and CBF4 that resulted in drought/salt/cold
tolerance in Arabidopsis (Jaglo-Ottosen et al., 1998, Science 280:
104-106). Since then, the orthologous genes of CBF/DREB have been
identified in many crop plants and functional tests revealed
conservation of function (reviewed in Xu et al., 2011. J. Int.
Plant Biol. 53: 570-585). It has also been shown that ectopic
overexpression of these TF genes caused, in addition to increased
stress tolerance, some specific phenotypic changes--dark-green,
dwarfed plants with higher levels of soluble sugars and proline
have been obtained. More recent evidence suggested the role of an
AP2 family protein SHINE/WAX INDUCER 1 (SHN) as a global level
regulator of cell wall biosynthesis which could be economically
valuable for biofuel production from lignocellulosic crops
(Ambavaram et al., 2011, Plant Physiol. 155: 916-931).
In studies with model plants, it has been shown that transcription
factors belonging to the AP2/ERF, NF-Y, bZIP, MYB, Zinc-finger and
NAC families confer tolerance to both biotic and abiotic stresses.
Comparative genomics has also been used to find genes with
conserved functions between model plants (mainly Arabidopsis) and
crop plants, such as rice and maize demonstrating the utility of
using the dicot-monocot models together. For example, expression of
an Arabidopsis AP2/ERF-like transcription factor in rice resulted
in an increase in leaf biomass and bundle sheath cells that
probably contributed to the enhanced photosynthetic assimilation
and efficiency (Karaba et al., 2009, Proc. Natl. Acad. Sci. USA
104: 15270-15275).
2. Reporter Genes and Selectable Marker Genes
Reporter genes or selectable marker genes may be included in an
expression cassette as described in US Patent Applications
20100229256 and 20120060413 incorporated by reference herein. An
expression cassette including a promoter sequence operably linked
to a heterologous nucleotide sequence of interest can be used to
transform any plant by any of the methods described above. Useful
selectable marker genes and methods of selection transgenic lines
for a range of different crop species are described in the examples
herein.
E. Transgene Expression in Plants
Plant promoters can be selected to control the expression of the
transgene in different plant tissues or organelles for all of which
methods are known to those skilled in the art (Gasser & Fraley,
1989, Science 244: 1293-1299). In one embodiment, promoters are
selected from those of eukaryotic or synthetic origin that are
known to yield high levels of expression in plant and algae. In a
preferred embodiment, promoters are selected from those that are
known to provide high levels of expression in monocots.
1. Inducible Promoters
Chemical-regulated promoters can be used to modulate the expression
of a gene in a plant through the application of an exogenous
chemical regulator. Depending upon the objective, the promoter may
be a chemical-inducible promoter, where application of the chemical
induces gene expression, or a chemical-repressible promoter, where
application of the chemical represses gene expression.
Chemical-inducible promoters are known in the art and include, but
are not limited to, the maize 1n2-2 promoter, which is activated by
benzenesulfonamide herbicide safeners, the maize GST promoter,
which is activated by hydrophobic electrophilic compounds that are
used as pre-emergent herbicides, and the tobacco PR-1 promoter
which is activated by salicylic acid. Other chemical-regulated
promoters include steroid-responsive promoters [see, for example,
the glucocorticoid-inducible promoter (Schena et al., 1991, Proc.
Natl. Acad. Sci. USA 88: 10421-10425; McNellis et al., 1998, Plant
J. 14: 247-257) and tetracycline-inducible and
tetracycline-repressible promoters (see, for example, Gatz et al.,
1991, Mol. Gen. Genet. 227: 229-237; U.S. Pat. Nos. 5,814,618 and
5,789,156, herein incorporated by reference in their entirety).
A three-component osmotically inducible expression system suitable
for plant metabolic engineering has recently been reported (Feng et
al., 2011, PLoS ONE 6: 1-9).
2. Constitutive Promoters
Constitutive promoters include, for example, the core promoter of
the Rsyn7 promoter and other constitutive promoters disclosed in WO
99/43838 and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter
(Odell et al., 1985, Nature 313: 810-812), rice actin (McElroy et
al., 1990, Plant Cell 2: 163-171), ubiquitin (Christensen et al.,
1989, Plant Mol. Biol. 12: 619-632; Christensen et al., 1992, Plant
Mol. Biol. 18: 675-689), pEMU (Last et al., 1991, Theor. Appl.
Genet. 81: 581-588), MAS (Velten et al., 1984, EMBO J. 3:
2723-2730), and ALS promoter (U.S. Pat. No. 5,659,026). Other
constitutive promoters are described in U.S. Pat. Nos. 5,608,149;
5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;
and 5,608,142.
3. Weak Promoters
Where low level expression is desired, weak promoters may be used.
Generally, the term "weak promoter" is intended to describe a
promoter that drives expression of a coding sequence at a low
level. Where a promoter is expressed at unacceptably high levels,
portions of the promoter sequence can be deleted or modified to
decrease expression levels. Such weak constitutive promoters
include, for example, the core promoter of the Rsyn7 promoter (WO
99/43838 and U.S. Pat. No. 6,072,050).
4. Tissue Specific Promoters
"Tissue-preferred" promoters can be used to target gene expression
within a particular tissue. Compared to chemically inducible
systems, developmentally and spatially regulated stimuli are less
dependent on penetration of external factors into plant sells.
Tissue-preferred promoters include those described by Van Ex et
al., 2009, Plant Cell Rep. 28: 1509-1520; Yamamoto et al., 1997,
Plant J. 12: 255-265; Kawamata et al., 1997, Plant Cell Physiol.
38: 792-803; Hansen et al., 1997, Mol. Gen. Genet. 254: 337-343;
Russell et al., 199), Transgenic Res. 6: 157-168; Rinehart et al.,
1996, Plant Physiol. 112: 1331-1341; Van Camp et al., 1996, Plant
Physiol. 112: 525-535; Canevascini et al., 1996, Plant Physiol.
112: 513-524; Yamamoto et al., 1994, Plant Cell Physiol. 35:
773-778; Lam, 1994, Results Probl. Cell Differ. 20: 181-196, Orozco
et al., 1993, Plant Mol. Biol. 23: 1129-1138; Matsuoka et al.,
1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590, and Guevara-Garcia
et al., 1993, Plant J. 4: 495-505. Such promoters can be modified,
if necessary, for weak expression.
4.i. Seed/Embryo Specific Promoters
"Seed-preferred" promoters include both "seed-specific" promoters
(those promoters active during seed development such as promoters
of seed storage proteins) as well as "seed-germinating" promoters
(those promoters active during seed germination). See Thompson et
al., 1989, BioEssays 10: 108-113, herein incorporated by reference.
Such seed-preferred promoters include, but are not limited to, Cim1
(cytokinin-induced message), cZ19B1 (maize 19 kDa zein), mi1ps
(myo-inositol-1-phosphate synthase), and celA (cellulose synthase).
Gamma-zein is a preferred endosperm-specific promoter. Glob-1 is a
preferred embryo-specific promoter. For dicots, seed-specific
promoters include, but are not limited to, bean .beta.-phaseolin,
napin, .beta.-conglycinin, soybean lectin, cruciferin, and the
like. For monocots, seed-specific promoters include, but are not
limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein,
waxy, shrunken 1, shrunken 2, and globulin 1. The stage specific
developmental promoter of the late embryogenesis abundant protein
gene LEA has successfully been used to drive a recombination system
for excision-mediated expression of a lethal gene at late
embryogenesis stages in the seed terminator technology (U.S. Pat.
No. 5,723,765 to Oliver et al.).
4.ii. Leaf Specific Promoters
Leaf-specific promoters are known in the art. See, for example,
WO/2011/041499 and U. S. Patent No 2011/0179511 A1 to Thilmony et
al.; Yamamoto et al., 1997, Plant J. 12: 255-265; Kwon et al.,
1994, Plant Physiol. 105: 357-367; Yamamoto et al., 1994, Plant
Cell Physiol. 35: 773-778; Gotor et al., 1993, Plant J. 3: 509-518;
Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138, and Matsuoka
et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590.
4.iii. Temporal Specific Promoters
Also contemplated are temporal promoters that can be utilized
during the developmental time frame, for example, switched on after
plant reaches maturity in leaf to enhance carbon flow.
4iv. Anther/Pollen Specific Promoters
Numerous genes specifically expressed in anthers and/or pollen have
been identified and their functions in pollen development and
fertility have been characterized. The specificity of these genes
has been found to be regulated mainly by their promoters at the
transcription level (Ariizumi et al., 2002, Plant Cell Rep. 21:
90-96 and references therein). A large number of anther- and/or
pollen-specific promoters and their key cis-elements from different
plant species have been isolated and functionally analyzed.
4.v. Floral Specific Promoters
Floral-preferred promoters include, but are not limited to, CHS
(Liu et al., 2011, Plant Cell Rep. 30: 2187-2194), OsMADS45 (Bai et
al., 2008, Transgenic Res. 17: 1035-1043), PSC (Liu et al., 2008,
Plant Cell Rep. 27: 995-1004), LEAFY, AGAMOUS, and AP1 (Van Ex et
al., 2009, Plant Cell Rep. 28: 1509-1520), AP1 (Verweire et al.,
2007, Plant Physiol. 145: 1220-1231), PtAGIP (Yang et al., 2011,
Plant Mol. Biol. Rep. 29: 162-170), Lem1 (Somleva & Blechl,
2005, Cereal Res. Comm. 33: 665-671; Skadsen et al., 2002, Plant
Mol. Biol. 45: 545-555), Lem2 (Abebe et al., 2005, Plant
Biotechnol. J. 4: 35-44), AGL6 and AGL13 (Schauer et al., 2009,
Plant J. 59: 987-1000).
4.vi. Combinations of Promoters
Certain embodiments use transgenic plants or plant cells having
multi-gene expression constructs harboring more than one promoter.
The promoters can be the same or different.
Any of the described promoters can be used to control the
expression of one or more of the transcription factor genes of the
invention, their homologs and/or orthologs as well as any other
genes of interest in a defined spatiotemporal manner.
F. Requirements for Construction of Plant Expression Cassettes
Nucleic acid sequences intended for expression in transgenic plants
are first assembled in expression cassettes behind a suitable
promoter active in plants. The expression cassettes may also
include any further sequences required or selected for the
expression of the transgene. Such sequences include, but are not
restricted to, transcription terminators, extraneous sequences to
enhance expression such as introns, vital sequences, and sequences
intended for the targeting of the gene product to specific
organelles and cell compartments. These expression cassettes can
then be transferred to the plant transformation vectors described
infra. The following is a description of various components of
typical expression cassettes.
1. Transcriptional Terminators
A variety of transcriptional terminators are available for use in
expression cassettes. These are responsible for the termination of
transcription beyond the transgene and the correct polyadenylation
of the transcripts. Appropriate transcriptional terminators are
those that are known to function in plants and include the CaMV 35S
terminator, the tm1 terminator, the nopaline synthase terminator
and the pea rbcS E9 terminator. These are used in both
monocotyledonous and dicotyledonous plants.
2. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression from
within the transcriptional unit and these sequences can be used in
conjunction with the genes to increase their expression in
transgenic plants. For example, various intron sequences such as
introns of the maize Adh1 gene have been shown to enhance
expression, particularly in monocotyledonous cells. In addition, a
number of non-translated leader sequences derived from viruses are
also known to enhance expression, and these are particularly
effective in dicotyledonous cells.
G. Coding Sequence Optimization
The coding sequence of the selected gene may be genetically
engineered by altering the coding sequence for optimal expression
in the crop species of interest. Methods for modifying coding
sequences to achieve optimal expression in a particular crop
species are well known (Perlak et al., 1991, Proc. Natl. Acad. Sci.
USA 88: 3324 and Koziel et al., 1993, Biotechnology 11:
194-200).
H. Construction of Plant Transformation Vectors
Numerous vectors available for plant transformation are known to
those of ordinary skill in the plant transformation arts. The genes
pertinent to this disclosure can be used in conjunction with any
such vectors. The choice of vector depends upon the selected
transformation technique and the target species.
Many vectors are available for transformation using Agrobacterium
tumefaciens. These typically carry at least one T-DNA sequence and
include vectors such as pBIN19. Typical vectors suitable for
Agrobacterium transformation include the binary vectors pCIB200 and
pCIB2001, as well as the binary vector pCIB 10 and hygromycin
selection derivatives thereof. (See, for example, U.S. Pat. No.
5,639,949).
Transformation without the use of Agrobacterium tumefaciens
circumvents the requirement for T-DNA sequences in the chosen
transformation vector and consequently vectors lacking these
sequences are utilized in addition to vectors such as the ones
described above which contain T-DNA sequences. The choice of vector
for transformation techniques that do not rely on Agrobacterium
depends largely on the preferred selection for the species being
transformed. Typical vectors suitable for non-Agrobacterium
transformation include pCIB3064, pSOG 19, and pSOG35. (See, for
example, U.S. Pat. No. 5,639,949).
I. Transformation and Selection of Cultures and Plants
Plant cultures can be transformed and selected using one or more of
the methods described above which are well known to those skilled
in the art. In switchgrass, selection occurs by incubating the
cultures on a callus growth medium containing bialaphos. In an
alternative embodiment, selection can occur in the presence of
hygromycin. Resistant calluses are then cultured on a regeneration
medium (Somleva, 2006, Agrobacterium Protocols, Wang K., ed., Vol.
2, pp 65-74, Humana Press; Somleva et al., 2002, Crop Sci. 42:
2080-2087) containing the preferred selection agent. Examples of
specific selectable markers and transgenic plant selection methods
for a number of crop species are described in the examples
herein.
EXAMPLES
Example 1
Identification and Functional Characterization of Candidate
Transcription Factor Genes Potentially Involved in Photosynthesis
and Biomass Related Traits
The following approaches were used to identify and annotate
potential switchgrass transcription factors (TFs):
A. Gene Prediction Based on Systems Biology Approach
A rice regulatory association network that has been developed based
on genome wide expression profiles (Ambavaram et al., 2011, Plant
Physiol. 155: 916-931) was used to identify switchgrass orthologs
of TFs with predicted function in the regulation of genes involved
in photosynthesis and biomass related traits. Publicly available
databases were used to perform BlastN and BlastP reciprocal
searches between the genomes of rice (a C.sub.3 monocot; website:
rice.plantbiology.msu.edu), maize (a monocot possessing the NADP-ME
subtype of C.sub.4 photosynthesis; found at world wide web
maizesequence.org and switchgrass an NAD-ME C.sub.4 monocot at
phytozome.net/search.php?show=blast&org=Org_Pvirgatum to
identify candidate genes for functional validation and experimental
analysis. Comparisons of gene ontology (GO) terms from the
molecular function category revealed the most obvious functions of
DNA binding and transcriptional regulatory activity of the
identified TFs.
Based on genome-wide orthologous prediction, candidate genes were
retrieved from the corresponding websites and their percentage of
identity was evaluated (TABLE 1).
TABLE-US-00001 TABLE 1 Candidate transcription factor genes. Rice
gene Maize gene Switchgrass ortholog % identity E-value
LOC_Os02g10480 GRMZM2G138349 Pavirv00027905m 87.75 1e-86
LOC_Os07g41580 GRMZM2G384528 Pavirv00029298m 94.83 4e-58
LOC_Os02g52670 GRMZM2G103085 Pavirv00031839m 78.00 3e-11
LOC_Os09g11480 EU942421 Pavirv00046166m 75.44 2e-13 LOC_Os03g09170
GRMZM2G113060 Pavirv00021049m 61.11 3e-37 LOC_Os02g32140
GRMZM2G016434 Pavirv00013751m 97.26 8e-27 LOC_Os09g29960
GRMZM2G089850 Pavirv00059600m 94.59 3e-17 LOC_Os11g06770
GRMZM2G544539 Pavirv00009307m 91.94 1e-19 LOC_Os04g52090
GRMZM2G068967 Pavirv00015875m 98.31 2e-21 LOC_Os04g55520
GRMZM2G119865 Pavirv00033364m 66.67 4e-18
B. Functional Annotation of Select Switchgrass TFs
According to the plant transcription factor database (see world
wide web at planttfdb.cbi.edu.cn) and switchgrass genome (world
wide web at phytozome.net), SEQ ID NO: 1 (Pavirv00046166m) and SEQ
ID NO: 2 (Pavirv00013751m) are switchgrass transcription factors
belonging to the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF)
family and SEQ ID NO: 3 (Pavirv00029298m) is a switchgrass
transcription factor from the Nuclear-Factor Y (NF-YB) family. The
analysis of their protein sequences using a database of protein
domains, families and functional sites (world wide web at
expacy.org) revealed the characteristic AP2 domain (SEQ ID NO: 4
and SEQ ID NO: 5, underlined) and NFYA-HAP2 motif (SEQ ID NO: 6,
underlined), respectively. Comparisons of gene ontology terms for
the switchgrass genes SEQ ID NO: 1 and SEQ ID NO: 2 revealed the
`transcription factor` activity (GO: 0003700), whereas SEQ ID NO: 3
belongs to the MNFs based on its sequence-specific transcription
regulator activity (GO: 00030528). According to the TF-function
association network, these switchgrass orthologous TF genes may be
associated with functions in "primary" carbon metabolism and
several "cellular metabolic" processes.
C. Expression Analysis of Novel Transcription Factors in
Switchgrass.
For validation of the bioinformatics findings, the tissue specific
expression of the candidate TF genes (TABLE 1) in switchgrass was
analyzed by RT-PCR. Total RNA was isolated from root (R), culm (C),
leaf sheath (LS), young leaf (YL), mature leaf (ML), and panicle
(P) tissues of wild type plants. After DNase treatment and column
purification, total RNA (200 ng per reaction) was subjected to
reverse transcription and PCR in a one-step RT-PCR assay (Qiagen)
with gene-specific primers.
The results revealed the differences in the expression levels of
the candidate TF genes (listed in TABLE 1) in young and mature
leaves, roots, and stem tissues (culm, leaf sheath and panicle).
Based on their expression patterns we identified three genes which
were highly expressed in mature leaf and these, three genes (SEQ ID
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) were selected for
overexpression and functional analysis in switchgrass. The highest
transcript accumulation for these three genes was observed in
mature leaves (FIG. 2). No expression of the selected AP2/ERF
transcription factors (SEQ ID NO: 1 and SEQ ID NO: 2) was detected
in roots under the experimental conditions, while transcripts of
the switchgrass NF-Y gene (SEQ ID NO: 3) were present at different
levels in all tissues analyzed (FIG. 2).
Based on the effects of these TFs on plant metabolism and phenotype
(see Example 5), the genes and the encoded polypeptides were
designated as PvSTR1 (STarch Regulator 1; SEQ ID NO: 1 and SEQ ID
NO: 4), PvSTIF1 (STress Inducible Factor 1; SEQ ID NO: 2 and SEQ ID
NO: 5), and PvBMY1 (BioMass Yield 1, SEQ ID NO: 3 and SEQ ID NO:
6).
D. Identification of Homologous Genes of PvSTR1, PvSTIF1 and PvBMY1
Transcription Factors:
The sequence homology search was performed by comparing the deduced
amino acid sequences of PvSTR1, PvSTIF1 and PvBMY1 to a translated
non-redundant nucleotide database found on the world wide web at
blast.ncbi.nlm.nih.gov and phytozome.net using tBLASTN and to a
protein database using BLASTP. Transcription factor genes that are
homologous to the transcription factors of the invention will
typically have a polypeptide sequence of their conserved domain or
the entire coding region 80% or more identical to the SEQ ID NOs:
4-6. As used herein, a "homolog" means a protein that performs the
same biological function as another protein including these
identified by sequence identity search. In silico analysis resulted
in the identification of several homologs of each of the three
transcription factors of the invention indicated as PvSTR2-5 (SEQ
ID NOs: 7-10), PvSTIF2-4 (SEQ ID NOs: 11-13), and PvBMY2-6 (SEQ ID
NOs: 14-18) for the homologs of PvSTR1, PvSTIF1 and PvBMY1,
respectively.
The copy number of each of the TF genes in the switchgrass genome
was also determined by Southern blot hybridizations. Two genotypes
from the switchgrass cultivar Alamo--56 and 16 (our designation)
were studied. Callus cultures from these genotypes were used in all
the experiments on switchgrass transformation (as described in
Example 3). The results revealed the presence of the same number of
homologs of PvSTR1, PvSTIF1 and PvBMY1 in the two genotypes
analyzed (FIG. 3).
Based on the existing sequential similarity, including the presence
of identical DNA-binding domains, overexpression of the identified
homologous genes PvSTR2-5, PvSTIF2-4, and PvBMY2-6 can readily be
tested for trait modifications similar to the ones induced by
PvSTR1, PvSTIF1 and PvBMY1.
E. Identification of Orthologous Genes of PvSTR1, PvSTIF1 and
PvBMY1 Transcription Factors:
"Orthologs" and "paralogs" refer to polynucleotide and polypeptide
sequences which are homologous to the claimed sequences. These
genes are related because they originate from a common ancestral
gene and potentially retain a similar function in the course of
evolution. Orthologs are structurally related genes in different
species that are derived by speciation, while paralogs are
structurally related genes in the same species that are derived by
genetic duplication. Orthologous genes are identified based upon
percentage similarity or identity of the complete sequence or of a
conserved domain. Closely related transcription factors can share
about 70%, 75%, or about 80% or more amino acid sequence identity.
Sequences with sufficient similarity may also bind to the same DNA
binding sites of transcriptional regulatory elements.
Orthologs of the switchgrass transcription factor genes PvSTR1,
PvSTIF1 and PvBMY1 were identified using methods well known in the
art. Orthologous polypeptide sequences from different plant species
with more than 75%, 80%, 85%, greater than 90% identity of the
conserved binding domains are shown in FIG. 4. The phylogenetic
relationships were also estimated based on the conserved domain
sequences of PvSTR1, PvSTIF1 and PvBMY1 (FIG. 5).
Example 2
Design and Construction of Transformation Vectors for
Overexpression of Transcription Factor Genes in Switchgrass.
All gene constructs were made using widely available genetic
components and standard molecular biology techniques. Each of the
genes was cloned in an individual expression cassette and 2-5
cassettes were assembled in one vector for plant
transformation.
Two sets of gene constructs, one set containing the bar gene
(conferring resistance to bialaphos) as a selectable marker and
another one with the hptII gene (conferring resistance to
hygromycin), were created for overexpression of the transcription
factor genes of the invention in switchgrass (TABLE 2, FIG. 6, FIG.
7).
TABLE-US-00002 TABLE 2 Summary of plant transformation vectors for
expression of transcription factors and PHB biosynthesis genes.
Gene of Marker Vector Locus Name interest.sup.1 gene.sup.2 pMBXS809
Pavirv00046166m PvSTR1 bar pMBXS810 Pavirv00013751m PvSTIF1 bar
pMBXS855 Pavirv00029298m PvBMY1 bar pMBXS881 Pavirv00046166m PvSTR1
hptII pMBXS882 Pavirv00013751m PvSTIF1 hptII pMBXS883
Pavirv00029298m PvBMY1 hptII .sup.1Driven by the maize cab-m5
promoter fused to the maize hsp70 intron; .sup.2Driven by the 35S
promoter.
The vectors pMBXS809, pMBXS810, and pMBXS855 (FIG. 6) were used for
Agrobacterium-mediated transformation of switchgrass for generation
of transgenic lines for functional analyses of the novel
transcription factors (see Example 3). In each vector, the
transcription factor gene is under the control of the cab-m5
light-inducible promoter of the chlorophyll a/b-binding protein in
maize (Sullivan et al., 1989, Mol. Gen. Genet. 215: 431-440; Becker
et al., 1992, Plant Mol. Biol. 20: 49-60) fused to the heat shock
protein 70 (hsp70) intron (U.S. Pat. No. 5,593,874), while the
marker genes are driven by the 35S promoter (TABLE 2).
The annotation of the genes and genetic elements assembled in the
vectors pMBXS809, pMBXS810, and pMBXS855 are presented in TABLE 3
(see also FIG. 6 and FIG. 7).
TABLE-US-00003 TABLE 3 Plant transformation vectors for
overexpression of the transcription factor genes PvSTR1, PvSTIF1
and PvBMY1 in switchgrass. Vector ID* TF gene/marker Annotation SEQ
ID Coordinates (bp) pMBXS809 PvSTR1/bar Agrobacterium T-DNA right
border 19 1 to 26 Cab-m5 promoter with hsp70 8951 to 10645 intron
to drive PvSTR1 gene PvSTR1 coding region 10646 to 11636 nos
terminator 11637 to 11891 CaMV35S promoter to drive bar gene 7911
to 8680 bar coding region 6543 to 7094 CaMV35S polyA terminator
6335 to 6537 Agrobacterium T-DNA left border 6260 to 6285 pMBXS810
PvSTIF1/bar Agrobacterium T-DNA right border 20 1 to 26 Cab-m5
promoter with hsp70 8951 to 10645 intron to drive PvSTIF1 gene
PvSTIF1 coding region 10646 to 11240 nos terminator 11241 to 11495
CaMV35S promoter to drive bar gene 7911 to 8680 bar coding region
6543 to 7094 CaMV35S polyA terminator 6335 to 6537 Agrobacterium
T-DNA left border 6260 to 6285 pMBXS855 PvBMY1/bar Agrobacterium
T-DNA right border 21 1 to 26 Cab-m5 promoter with hsp70 8951 to
10645 intron to drive PvBMY1 gene PvBMY1 coding region 10646 to
11961 nos terminator 11978 to 12232 CaMV35S promoter to drive 7911
to 8680 bar gene bar coding region 6543 to 7094 CaMV35S polyA
terminator 6335 to 6537 Agrobacterium T-DNA left border 6260 to
6285 *All vectors are based on the transformation vector
pCambia3300 found at world wide web at cambia.org; the bar gene
(conferring resistance to bialaphos) is used as a marker for
selection of transformed callus cultures and plants.
Example 3
Transformation of Switchgrass
Highly embryogenic callus cultures initiated from different
explants were used for introduction of the gene constructs
described in Example 2.
Culture Initiation and Plant Regeneration:
Callus cultures were initiated from mature caryopses of cv. Alamo
following a previously published procedure (Denchev & Conger,
1994, Crop Sci., 34: 1623-1627). Their embryogenic potential and
plant regeneration ability were evaluated as described previously
(U.S. Pat. No. 8,487,159 to Somleva et al.).
Switchgrass plants from Alamo genotype 56 (Somleva et al., 2008,
Plant Biotechnol. J. 6: 663-678; U.S. Pat. No. 8,487,159 to Somleva
et al.) grown under greenhouse conditions were used for initiation
of immature inflorescence-derived callus cultures. The top culm
nodes of elongating tillers with 3-4 visible nodes were used for
development of inflorescences in tissue culture following a
previously published procedure (Alexandrova et al., 1996, Crop Sci.
36: 175-178). Callus cultures were initiated from individual
spikelets from in vitro developed panicles and propagated by
transferring on to a fresh medium for callus growth (Denchev and
Conger, 1994, Crop Sci. 34: 1623-1627) every four weeks.
Callus cultures were grown at 27.degree. C., in the dark and
maintained by monthly subcultures on a fresh medium for callus
growth (Somleva et al., 2002, Crop Sci. 42: 2080-2087). For plant
regeneration, calluses were plated on MS basal medium supplemented
with 1.4 .mu.M gibberellic acid and incubated at 27.degree. C. with
a 16-h photoperiod (cool white fluorescent bulbs, 80
.mu.mol/m.sup.2/s).
Transformation of Mature Caryopsis- and Immature
Inflorescence-Derived Cultures:
Highly embryogenic callus cultures were transformed with
Agrobacterium tumefaciens following previously published protocols
(Somleva et al., 2002, Crop Sci. 42: 2080-2087; Somleva, 2006,
Agrobacterium Protocols, Wang K., ed., pp 65-74: Humana Press).
Transformed cultures and plants regenerated from them were selected
with 200 mg/L hygromycin (WO 2010/102220 A1 and US 2010/0229256 A1
to Somleva & Ali) or 10 mg/L bialaphos (Somleva et al., 2002,
Crop Sci. 42: 2080-2087; Somleva, 2006, Agrobacterium Protocols,
Wang K., ed., pp 65-74: Humana Press). Transgenic plants
overexpressing the transcription factor genes PvSTR1, PvSTIF1, and
PvBMY1 were obtained from cultures transformed with the vectors
pMBXS809, pMBXS810, and pMBXS855 (TABLE 2). The presence of the
transcription factor and marker genes in putative transformants was
confirmed by PCR using primers specific for the coding regions of
the transgenes and the amplification conditions described
previously (Somleva et al., Plant Biotechnol. J. 6: 663-678). More
than 200 T.sub.0 plants representing 58 independent transformation
events were identified (TABLE 4). Plants regenerated from
untransformed callus cultures and grown under the same conditions
were used as controls (non-transgenic plants; wild-type plants) in
expression and functional analyses of transgenic lines.
TABLE-US-00004 TABLE 4 Transformations for overexpression of
transcription factors in switchgrass. Gene of Alamo genotype
56.sup.1 Alamo genotype 16.sup.2 Vector interest # events.sup.3 #
plants.sup.4 # events.sup.3 # plants.sup.4 pMBXS809 PvSTR1 8 60 6
31 pMBXS810 PvSTIF1 14 44 12 60 pMBXS855 PvBMY1 9 27 9 14 Total: 31
111 27 105 .sup.1immature inflorescence-derived callus cultures
from this genotype were transformed; .sup.2mature caryopsis-derived
callus cultures from this genotype were transformed; .sup.3number
of bialaphos-resistant callus lines producing at least one
transgenic plant; .sup.4number of primary transformants (as
confirmed by PCR).
After transfer to soil, transgenic and wild-type plants obtained
from different transformation experiments were grown in a
greenhouse at 27.degree. C./24.degree. C. (day/night) with
supplemental lighting (16-h photoperiod, sodium halide lamps).
Example 4
Expression Analyses of Transgenic Switchgrass Plants Transformed
with the Genes Encoding the Transcription Factors PvSTR1, PvSTIF1,
and PvBMY1
In all experiments, total RNA was isolated from the second youngest
leaf of primary transformants and control wild-type plants (3
plants per line) prior to transfer to soil using RNeasy Plant Mini
Kit (Qiagen). After DNase treatment and column purification,
different amounts of RNA were used for RT-PCR and qRT-PCR
(quantitative reverse transcription polymerase chain reaction or
real-time RT-PCR). Quantitative analysis of the differences in the
expression levels of the TF genes in transgenic and control lines
was performed by qRT-PCR using .beta.-actin as a reference. For
each sample, 500 ng of total RNA was converted into cDNA using
iScript cDNA synthesis kit (Bio-Rad). The cDNA was diluted and
subjected to real-time PCR using Fast SYBR.RTM. Green Master Mix
(Life Technologies) in an Applied Biosystems 7500 Fast Real-Time
PCR system. The amplification curves for each line were generated
and used to calculate the relative expression ratio (fold change)
compared to the wild type control. All of the transgenic lines
analyzed showed significantly higher levels of expression of the
transcription factor genes in the transgenic lines as compared to
the control plants transcript accumulation (from 3 to 9.5 times
higher as shown in FIG. 8).
Example 5
Effects of the Overexpression of the Transcription Factors PvSTR1,
PvSTIF1 and PvBMY1 on Biomass Production and Photosynthetic
Activity in Transgenic Switchgrass Plants
For functional characterization of PvSTR1, PvSTIF1 and PvBMY1
transcription factors, biochemical and physiological analyses were
performed with transgenic and control wild-type switchgrass plants
grown in soil for two months. Both groups of plants were from two
Alamo genotypes--56 and 16 (our designation) differing in their
morphology.
Measurements of Photosynthetic Activity:
For analyses of the photosynthesis rate in plants overexpressing
the TF genes of the invention, various parameters were measured in
light adapted leaves using a Dual-PAM-100 Measuring System (Heinz
Walz GmbH). All measurements were performed with the leaf attached
to the second node from the base of vegetative tillers with the
forth emerging leaf.
The functioning of photosystem I (PSI) and photosystem II (PSII)
was studied in terms of photochemical quantum yield (Y) and
electron transport rate (ETR). Transgenic lines with improved
photosynthetic capacity compared to wild type controls from the
corresponding genotypes were identified (results are summarized in
TABLE 5 for PvSTR1, PvSTIF1 and PvBMY1 lines, respectively). In
some of the transgenic plants analyzed, the quantum yield of PSI
and PSII were significantly increased at photosynthetically active
radiation (PAR) of 30-37 .mu.mol m.sup.-2 s.sup.-1 (TABLE 5). The
electron transport rates of PSI and PSII in some of the transgenic
plants were significantly elevated compared to the wild type
control plants at PAR.gtoreq.119 .mu.mol m.sup.-2 s.sup.-1 (TABLE
5).
TABLE-US-00005 TABLE 5 Effect of the overexpression of
transcription factors on photosynthesis. Y(I) Y(II) ETR(I) ETR(II)
Max % to Max % to Max % to Max % to TF gene value.sup.1
control.sup.2 value.sup.1 control.sup.2 value.sup.1 co- ntrol.sup.2
value.sup.1 control.sup.2 PvSTR1 0.802 137 0.735 112 46.8 131 12.2
130 PvSTIF1 0.746 125 0.714 108 49.7 139 12.8 136 PvBMY1 0.887 148
0.722 110 48.5 136 13.2 140 .sup.1The maximum value measured in
individual transgenic switchgrass plants; .sup.2Compared to the
average values (5-6 plants, 2-3 measurements per plant) measured in
the corresponding wild-type controls in terms of genotype, growth
period and sampling date; Abbreviations: Y(I), photochemical
quantum yield of photosystem I (PSI) - reflects the efficiency of
quantum energy absorption by PSI reaction centers; Y(II), effective
quantum yield of photosystem II (PSII) - represents the portion
(from 0 to 1) of absorbed quanta that is converted into chemically
fixed energy by the PSII reaction centers (the other portion of the
quanta is dissipated into heat and fluorescence); ETR(I), electron
transport rate of PSI - represents the rate of the cyclic or
non-cyclic transfer of electrons from the excited reaction-center
chlorophyll a molecule to the electron acceptor(s); ETR(II),
electron transport rate of PSII - reflects the efficiency of the
non-cyclic electron transfer.
Because of the linear correlation between the quantum yield of PSII
and CO.sub.2 fixation in C.sub.4 plants (Leipner et al., 1999,
Environ. Exp. Bot. 42: 129-139; Krall & Edwards, 1992, Physiol.
Plant. 86: 180-187), the data suggested that the overexpression of
the transcription factors resulted in improvement of the overall
rate of photosynthesis (TABLE 5). This suggestion was supported by
the significant increase in the electron transport rate (TABLE 5)
based on the linear correlation between photosynthesis rate and ETR
due to the lack of photorespiration in C.sub.4 species (Kakani et
al., 2008, Photosynthetica 46: 420-430). In addition, the enhanced
ETR of PSI in some of the transgenic lines (TABLE 5) could indicate
increased cyclic electron transport around PSI which provides the
additional ATP needed for the CO.sub.2 fixation cycle of the
C.sub.4 photosynthesis (Kiirats et al. 2010, Photosynth. Res. 105:
89-99).
After measurements of the photosynthetic activity, the leaf blades
were sampled and used for determination of the contents of primary
metabolites and photosynthetic pigments as well as for RNA and
protein isolation.
Primary Metabolites:
Leaf tissue was ground in liquid nitrogen and freeze-dried for 3
days. Resultant leaf powder was used for measurements of the levels
of primary metabolites using different analytical methods: a
quantitative, enzymatic assay for starch (Starch Assay Kit, Sigma)
and HPLC for soluble sugars and fatty acids.
The levels of products of the central carbon metabolism (starch,
sucrose, glucose, and fatty acids) were measured in more than 80
transgenic plants representing 30 independent lines (10 lines/TF
gene). The results are summarized in TABLE 6.
Photosynthetic Pigments:
Chlorophyll a, chlorophyll b, and carotenoids were determined in
freshly harvested leaf tissue following a previously described
procedure (Lichtenhaler, 1987, Methods Enzymol., 148: 350-382). The
experiments were performed with 97 transgenic plants representing
30 independent lines (10 lines/TF gene). The results are summarized
in TABLE 6.
This initial screening resulted in the identification of transgenic
lines (2-5 plants per line) accumulating primary metabolites and
pigments at levels significantly higher than the control
untransformed plants grown under the same conditions. The data
confirmed the predicted function of the tested TF genes as global
regulators of the central carbon metabolism (see Example 1) and
correlated with the results from the gene expression microarray
analysis (see Example 7).
TABLE-US-00006 TABLE 6 Summary of the results from screening of
transgenic switchgrass lines overexpressing the TF genes PvSTR1,
PvSTIF1, and PvBMY1. Metabolites Biomass Soluble Fatty Pigments Dry
No. of TF gene Line ID Starch sugars acids Chlorophyll Carotenoids
weight tillers PvSTR1 56-1 128 101 123 144 133 132 118 56-2 125 97
111 137 125 112 111 56-3 123 156 107 88 79 102 95 56-7 160 138 117
144 141 139 131 56-9 159 120 115 116 111 140 128 56-13 152 125 107
201 181 143 152 56-14 339 244 80 109 100 113 112 16-4 n.a. n.a. 85
93 80 91 114 16-5 113 129 89 104 78 n.a. n.a. 16-6 168 115 90 119
124 103 111 PvSTIF1 56-2 180 93 130 142 129 94 68 56-3 104 123 128
136 121 105 108 56-4 n.a. 86 128 158 134 98 107 56-8 223 73 122 163
141 107 102 16-1 184 119 91 136 142 131 120 16-2 222 101 84 135 134
116 112 16-3 134 105 89 115 126 n.a. n.a. 16-4 153 114 88 131 132
137 129 16-6 201 88 90 125 139 142 138 16-9 186 117 101 125 139 111
126 PvBMY1 56-1 97 96 106 113 113 120 149 56-4 174 100 n.a. 106 84
115 142 56-6 136 137 94 112 98 123 135 56-7 123 127 110 117 117 123
156 56-8 141 152 99 103 101 133 148 56-9 223 192 104 80 73 124 158
16-1 n.a. 104 71 79 n.a. n.a. n.a. 16-2 126 99 106 111 111 124 194
16-3 270 158 75 92 91 n.a. 79 16-5 109 71 81 103 122 99 88 Values
are average from measurements of 2-5 plants per transgenic line or
wild type and are presented as % to the corresponding wild-type
control in terms of genotype, growth period and sampling date;
n.a.--not analyzed.
Individual plants with significantly higher levels of starch
(4.2-fold increase), sucrose (4.4-fold increase), glucose (2.7-fold
increase), fatty acids (1.5-fold increase), and total chlorophyll
(2.5-fold increase) were identified (TABLE 7).
TABLE-US-00007 TABLE 7 Effect of the overexpression of
transcription factors on the levels of primary metabolites and
photosynthetic pigments in switchgrass leaves. Metabolite/ No. of
plants Max value % to TF gene Pigment analyzed measured.sup.1
control.sup.2 Starch 26 11.659 405 PvSTR1 Sucrose 27 5.150 331
Glucose 27 0.575 192 (10 lines; Total fatty acids 19 4.065 148 30
plants Chlorophyll a + b 28 2.337 203 in total) Carotenoids 28
0.335 187 Starch 38 5.558 415 PvSTIF1 Sucrose 38 2.681 165 Glucose
38 0.735 269 (10 lines; Total fatty acids 32 4.159 150 41 plants
Chlorophyll a + b 39 2.960 252 in total) Carotenoids 39 0.359 199
Starch 29 12.272 426 PvBMY1 Sucrose 27 6.768 435 Glucose 27 0.432
132 (10 lines; Total fatty acids 23 3.916 143 31 plants Chlorophyll
a + b 30 1.589 135 in total) Carotenoids 30 0.259 144 .sup.1Data
for starch, sucrose, glucose and fatty acids presented as % DW;
data for chlorophyll a + b and carotenoids presented as mg/g FW;
.sup.2Values compared to the corresponding wild-type control in
terms of genotype, growth period and sampling date.
A similar increase in the levels of primary metabolites was also
detected in other plant parts. For example, the starch content in
the second leaf of a plant from line 56-14 was 405% to the control
(TABLE 6 and TABLE 7). The third and flag leaves from this plant
also contained 4 times more starch than the corresponding leaves
from wild-type control plants.
Unexpectedly, some of the transgenic switchgrass plants with
significantly increased levels of starch and soluble sugars
produced the same or slightly higher amounts of biomass compared to
the control plants. For example, a plant from the PvBMY1 line 56-8
(TABLE 6) contained 3.2.times. more starch and 2.2.times. more
sucrose and glucose than the corresponding control plants but its
biomass was only 1% higher than the average biomass of the wild
type plants. The total biomass yield of the plant with the highest
starch content (415% to control) among the PvSTIF1 plants was
similar to the biomass of the control wild-type plants. A 20%
increase in biomass production was measured in a plant from the
PvSTR1 line 56-14 (TABLE 6) despite the fact that the content of
starch and soluble sugars in the leaves of this plant was 333% to
the control.
Protein Analyses:
Western blot analysis of total proteins was performed as described
previously (Somleva et al., 2008, Plant Biotechnol. J. 6: 663-678).
An increase in the abundance of the proteins of the light
harvesting centers of PSI (LhcA proteins) and PSII (LhcB proteins)
was detected in most of the PvSTR1 and PvSTIF1 lines analyzed
compared to the corresponding wild-type control (examples for LhcA3
and LhcB5 are shown in FIG. 9A). These findings are in agreement
with the enhanced chlorophyll content in these lines (TABLE 6 and
TABLE 7). The accumulation of phosphoenolpyruvate (PEPC) protein in
most of the PvBMY1 lines was higher than in the wild-type plants
(an example is shown in FIG. 9B).
This is the first report on the effect of any transcription factor
on the abundance of Lhc and PEPC proteins.
Biomass Accumulation and Plant Development:
The growth and development of transgenic switchgrass plants
overexpressing the transcription factors of the invention were
monitored in terms of plant height and number of tillers after
transfer to soil. All of the transgenic plants had larger leaf
blades and longer internodes compared to the wild type plants from
the corresponding genotype.
Total biomass yield was evaluated in plants grown under greenhouse
conditions for five months as described in publications, WO
2012/037324 A2 and US 2012/0060413 to Metabolix. All vegetative and
reproductive tillers at different developmental stages from each
plant were counted and cut below the basal node. Leaves and stem
tissues were separated, cut into smaller pieces, air-dried at
27.degree. C. for 12-14 days and dry weight measurements were
obtained. The number and ratio of vegetative to reproductive
tillers were evaluated to compare the developmental patterns of
transgenic and control plants.
The total biomass of 82 transgenic plants representing 29 TF lines
and 12 wild type plants was measured. Transgenic lines with
increased biomass yield (up to 142% to the control) and number of
tillers (up to 194% to the control) were obtained (TABLE 6).
Most of the transgenic plants--81.5% of the analyzed PvSTR1 plants,
66.7% of the PvSTIF1 plants, and 82.1% of the PvBMY1 plants had
higher biomass yield (up to 162%) compared to the control plants
(TABLE 8). TF-overexpressing plants with significantly increased
number of tillers (up to 216% to the control) were also
identified.
TABLE-US-00008 TABLE 8 Effect of the overexpression of
transcription factors on switchgrass biomass production. Max value
% to TF gene Biomass measured [g DW] control.sup.1 PvSTR1 Total
90.6 162 (10 lines; Leaves 25.9 149 27 plants Stem 71.4 184
analyzed) No. of tillers.sup.2 45 190 PvSTIF1 Total 70.9 153 (10
lines; Leaves 17.5 162 27 plants Stem 53.4 150 analyzed) No. of
tillers.sup.2 25 166 PvBMY1 Total 79.6 142 (9 lines; Leaves 25.2
145 28 plants Stem 56.8 146 analyzed) No. of tillers.sup.2 51 216
.sup.1Values compared to the corresponding wild-type control in
terms of genotype, growth period and sampling date; .sup.2Total
number of vegetative and reproductive tillers at different
developmental stages (emerging tillers not included).
Similar patterns in the biomass productivity were observed in
plants grown in soil for six months after repotting. For example, a
plant from line 16-6 whose biomass was 148.8% to the control 4
months after transfer to soil yielded about 300 g DW total biomass
after repotting which was 182.3% to the corresponding control.
Example 6
Evaluation of the Stress Response of Switchgrass Lines
Overexpressing Transcription Factors
To validate the role of the transcription factors of the invention
in improvement of plant stress tolerance, a novel method for
screening of large populations of transgenic and control plants for
their response to drought and salinity has been developed. It
utilizes the previously developed tissue culture-based technology
for propagation and improvement of polymer production in transgenic
switchgrass plants (WO 2010/102220 A1 and US 2010/0229256 A1 to
Somleva and Ali).
The stress-inducing conditions were established using
non-transformed, wild-type plants. Polyethylene glycol (PEG) and
NaCl were chosen for induction of drought and salinity stresses,
respectively. Hundreds of plants were regenerated from immature
inflorescence-derived callus cultures from Alamo genotype 56. After
3-4 weeks culture on MS medium for plant regeneration,
phenotypically uniform plants were transferred to larger tissue
culture containers containing the same medium supplemented with
different concentrations of PEG and NaCl. Since the first
stress-induced changes in plant morphology, such as leaf wilting
and yellowing were observed after 3-4 days of treatment in
preliminary experiments, this time period was used in the
subsequent experiments. The relative water content (RWC), levels of
photosynthetic pigments and abundance of the chloroplastic Cu--Zn
superoxide dismutase (SOD) protein were used as stress markers.
They were measured as follows: RWC according to Smart &
Bingham, 1974, Plant Physiol. 53: 258-260, pigments as described by
Lichtenhaler, 1987, Methods Enzymol., 148: 350-382 and SOD using a
Plant SOD ELISA kit (MyBioSource).
Three different concentrations of the stress inducing agents were
tested in 3 replicates each (10 plants/replicate). Based on the
results from these treatments, 200 mM NaCl and 15% PEG were used in
the experiments with the TF plants.
Plants regenerated from immature inflorescence-derived callus
cultures initiated from well characterized TF lines along with wild
type plants (regenerated from non-transformed cultures) were
subjected to stress-inducing treatments under the conditions
described above. Non-treated transgenic and wild type plants served
as controls. All treatments were conducted in 3-4 replicates (10
plants per replicate).
As shown in the example in FIG. 10, treatment with 200 mM NaCl
resulted in a slight decrease in RWC in the transgenic plants--2.2%
and 1.6% in PvSTR1 and PvSTIF1 plants, respectively, while RWC in
the wild-type plants was reduced with 7.6% compared to the
non-treated control (FIG. 10A). Interestingly, RWC in the
non-stressed TF plants was 4-8% higher than the relative water
content in the wild type plants. Non-treated transgenic plants
contained significantly lower amounts of the chloroplastic Cu--Zn
superoxide dismutase (SOD) protein (as determined by ELISA)
compared to non-stressed wild-type plants (FIG. 10B). High salinity
stress conditions induced a similar increase in SOD levels in the
PvSTIF1 and wild-type plants (16% and 19%, respectively) while the
SOD protein content detected in the PvSTR1 plants was with about 7%
higher than in the non-treated control (FIG. 10B). The non-treated
TF plants also contained higher levels of photosynthetic
pigments--27% and 43% higher total chlorophyll content in PvSTR1
and PvSTIF1 plants, respectively, compared to the unstressed wild
type plants (FIG. 10C). The salinity stress caused a significant
decrease (37-63%) in the chlorophyll content in both transgenic and
wild type plants. The content of carotenoids in the stressed wild
type plants was reduced with 18.2% compared to the non-treated
plants, while in the TF plants it was 30-39% lower than in the
corresponding control plants (FIG. 10C). Similar changes in the
stress markers were observed when the plants were subjected to
PEG-induced drought stress.
This is the first report demonstrating the effect of the
overexpression of the transcription factors of the invention on
plant stress response and the possibility to test the role of any
transcription factors in this process under in vitro
conditions.
Example 7
Global Gene Expression Analysis of Switchgrass Transgenic Lines
Overexpressing PvSTR1, PvSTIF1 and PvBMY1
To identify the genes whose regulation by the transcription factors
of the invention resulted in the observed improved biomass yield
and stress tolerance (Examples 5 and 6), gene expression profiling
was performed using an Affymetrix switchgrass cDNA GeneChip.
Gene expression microarrays, data processing and normalization:
Three of the best performing switchgrass lines overexpressing one
of the TF genes (TABLES 6-8) were selected for the microarray gene
expression analysis. Total RNA was isolated from the second leaf of
vegetative tillers (3-4 tillers per plant) as described in Example
3. RNA extracts from three plants from each line were pooled and
their quality was evaluated using RNA Nano Chip (Agilent
Technologies) according to the manufacturer's instructions. The
microarray analysis was conducted using an Affymetrix switchgrass
GeneChip containing probes to query approximately 43,344
transcripts following the manufacturer's protocol website:
affymetrix.com). Raw numeric values representing the signal of each
feature were imported into AffylmGUI and the data were background
corrected, normalized, and summarized using Robust Multiarray
Averaging (RMA). A linear model was used to average data between
the replicates and to detect differential expression. Data quality
was assessed using box and scatter plots to compare the intensity
distributions of all samples and to assess the gene expression
variation between the replicates, respectively. Genes with
significant probe sets (FDR<0.1) with .gtoreq.2.0-fold changes
compared to the corresponding wild-type controls were considered
differentially expressed.
Identification and functional annotations of differentially
expressed genes regulated by PvSTR1, PvSTIF1 and PvBMY1: Since the
genome sequence of switchgrass is not well annotated, a reciprocal
BLAST analysis (a common computational method for predicting
putative orthologs consisting of two subsequent sets of BLAST
analysis) was performed for functional annotation of the
differentially expressed genes and their corresponding orthologs.
The first BLAST was conducted using the well annotated whole genome
sequences of maize, sorghum, rice and Arabidopsis. BLASTN or
TBLASTX are generally used for analyses of a polynucleotide
sequence, while BLASTP or TBLASTN--for a polypeptide sequence. The
first set of BLAST results may optionally be filtered. The
full-length sequences of either the filtered results or
non-filtered results are then BLASTed back (second BLAST) against
sequences from the organism from which the query sequence is
derived. The results of the first and second BLASTs are then
compared. If this returns the switchgrass gene originally used as
the highest scorer, then the two genes are considered putative
orthologs.
The numbers of the annotated genes up- or down-regulated by PvSTR1,
PvSTIF1 and PvBMY1 in transgenic switchgrass plants are shown in
FIG. 11A. A further analysis of the gene expression data revealed
that 450, 135, and 619 genes were up-regulated (FIG. 11B) and 165,
164, and 231 genes were down-regulated (FIG. 11C) by PvSTR1,
PvSTIF1 and PvBMY1, respectively. Only 1-2 genes were commonly
regulated by all three TFs. A relatively small portion of the
differentially expressed genes regulated by PvSTIF1 was also
regulated by the other two TFs, while more than 280 genes were
regulated by both PvSTR1 and PvBMY1 (FIG. 11B-C).
These findings indicate that the transcription factors of the
invention regulate the expression of genes involved in key
processes and pathways by different mechanisms.
Downstream transcription factors regulated by PvSTR1, PvSTIF1 and
PvBMY1: Among the up-regulated genes identified by microarray
analysis of transgenic switchgrass lines, 80 were predicted to be
transcription factors based on the presence of a DNA-binding domain
(Plants TF database v. 3.0). Several of these homologous TF genes
have functionally been validated in model and crop plants as
regulators of genes involved in economically important agronomic
traits, such as biomass production, grain yield and abiotic stress
tolerance.
These results confirm that the transcription factors of the current
invention appear to function as global transcriptional regulators.
The number and variety of the transcription factor genes identified
by the microarray analysis indicate that PvSTR1, PvSTIF1 and PvBMY1
regulate key genes in several major pathways and their branches
either directly or through downstream transcription factors.
Pathway analysis of differentially expressed genes: For more
detailed analysis of the regulatory pattern of the transcription
factors of the invention, the differential expression data was used
for identification of metabolic and/or signaling pathways or
portions of a pathway up-regulated in transgenic switchgrass
plants. To investigate the biological functions of differentially
expressed genes, gene ontology (GO) analysis was performed to
identify the "biological processes category" using a publicly
available database website: bioinfo.cau.edu.cn/agriGO/index.php).
The results revealed that PvSTR1 and BMY1 significantly increased
the expression of several genes involved in primary metabolic
processes, such as photosynthesis and carbohydrate metabolism, and
in amino acid and cell wall biosynthesis related pathways, while
most of the genes up-regulated by PvSTIF1 were categorized as
transcription factors (FIG. 12).
Taken together, the results presented here and in Example 5
indicate that central carbon metabolism in the transgenic plants in
which the transcription factors have been over expressed results in
major global impact on central carbon metabolism.
Transcriptional regulatory network of the central carbon metabolism
in switchgrass: Central carbon metabolism (CCM) is crucial for
plant growth and development because of its key role in the
generation of accessible energy and primary building blocks for
other metabolic pathways. The gene expression analysis of
switchgrass lines overexpressing PvSTR1, PvSTIF1 and PvBMY1
revealed a distinctive up-regulation of several genes involved in
photosynthesis and carbohydrate metabolism as well as in the
primary metabolic processes, which are not only necessary for plant
growth and development but often confer highly desirable
traits.
Example 8
Transcription Factor-Mediated Modifications of Economically
Valuable Traits
The switchgrass transcription factors characterized in this
invention (SEQ ID NOs: 1-6) and their homologs (SEQ ID NOs: 7-18)
can be introduced into the genome of other plants, including but
not limited to the varieties of grain and forage cereals and
grasses, oilseeds, biomass crops, legumes, trees, and vegetables.
The orthologous genes identified in this invention (see Example 1)
can also be used for genetic engineering of economically important
crops and model plant systems. It is well known, that transcription
factor gene sequences are conserved across different species lines,
including plants (Goodrich et al., 1993, Cell 75:519-530; Lin et
al., 1991, Nature 353: 569-571). Since the sequences of the
switchgrass TFs STR1, STIF1, and BMY1 are related to sequences in
other plant species, one skilled in the art can expect that, when
expressed in other plants, the switchgrass TF genes and/or their
orthologs can have similar effects on plant metabolism and
phenotype to those demonstrated herein. For optimal results, both
sequential and phylogenetical analyses of the TF genes need to be
performed. Since sorghum (Sorghum bicolor L.) and maize (Zea mays
L.) are closely related to switchgrass (FIG. 5), both the
switchgrass genes and their corresponding orthologs (FIG. 4) can be
expressed to increase the photosynthetic activity, to up-regulate
the carbon and nitrogen metabolism as well as to improve the stress
tolerance of these crops resulting in higher biomass and/or grain
production. In sorghum, for example, PvSTR1, PvSTIF1 and PvBMY1 and
their respective orthologs with accession numbers XP002463183,
XP002452171 and XP002463163 (identified using the methods described
in Example 1) would be expected to have similar effects, while in
soybean (Glycine max L.), the orthologous genes (accession numbers
NP001238200, XP003522453 and XP003546199) would be preferred due to
the distant phylogenetic relation between this crop and switchgrass
(FIG. 5). In crops with unknown whole genome sequence, orthologous
genes from phylogenetically close species can be used. For example,
Arabidopsis orthologs of the transcription factors of the invention
can be expressed in camelina to achieve the desired trait
modification.
The coding sequences can be cloned in expression cassettes and
assembled in single- or multi-gene vectors using the methods
provided in the invention. Any of the methods for plant
transformation described herein can be used to introduce the TF
genes into the target plant. For example, particle bombardment with
whole plasmids or minimum cassettes can be used for gene delivery
to callus cultures initiated from immature zygotic embryos in wheat
(Okubara et al., 2002, Theor. Appl. Genet. 106: 74-83) and barley
(Wan & Lemaux, 1994, Plant Physiol. 104: 37-48) and to callus
induced from immature leaf rolls from sugarcane (Snyman et al.,
2006, Plant Cell Rep. 25: 1016-1023) and energy cane (Fouad et al.,
2009, In Vitro Cell. Dev. Biol. 45: S74). The expression of
switchgrass transcription factors and their orthologs can be
engineered by Agrobacterium-mediated transformation in different
crops, such as rice (Sahoo et al., 2011, Plant Methods 7:49-60),
other small grain crops (reviewed in Shrawat and Lorz, 2006, Plant
Biotech. J. 4: 575-603), industrial crops (cotton, Leelavathi et
al., 2004, Plant Cell Rep. 22: 465-470; tobacco, Horsch et al.,
1985, Science 227: 1229-1231) as well as crops with C.sub.4
photosynthesis, such as maize, sugarcane, sorghum, sweet sorghum,
and pearl millet (reviewed in Somleva et al., 2013, Plant Biotech.
J. 11: 233-252). The floral dip method can be used for
transformation of oilseed crops, such as canola (Li et al., 2010,
Int. J. Biol. 2: 127-131) and camelina (Liu et al., 2012, In Vitro
Cell. Dev. Biol. 48: 462-468). Both physical and biological
transformation methods have been developed for some crops (e.g.,
soybean, reviewed in Yamada et al., 2012, Breed. Sci. 61: 480-494)
and the more efficient method can be used for the purposes of this
invention.
Different promoters can be useful for controlling the expression of
the TFs of the invention depending on the crop and phenotype of
interest. Both constitutive and inducible promoters (responding to
environmental, chemical and hormonal signals) can be used. For
example, the maize light-inducible cab-m5 promoter is suitable for
engineering bioenergy crops, such as switchgrass (Somleva et al.,
2008, Plant Biotech. J. 6: 663-678) and sugarcane (Petrasovitch et
al., 2012, Plant Biotech J. 10: 569-578) because of its high
activity in leaf tissue.
Promoters capable of driving the expression of a TF gene in an
organ-specific and developmentally-regulated manner are of a
particular interest for modifications of economically valuable
traits. The engineered spatiotemporal activity of the transcription
factors of the invention can be useful, for example, for increased
grain yield in maize, rice, wheat, barley and grain varieties of
sorghum, for increased oil content in canola and camelina, and for
modifications of the biomass composition in bioenergy crops, such
as switchgrass, sugarcane, Miscanthus, sweet sorghum and energy
cane. The transcription factor genes of the invention, their
homologs and orthologs can be overexpressed in photosynthetic
tissues during different stages of embryo and seed development for
improvement of grain yield without increasing the production of
vegetative biomass. This approach requires the use of promoters
with high activity and tightly controlled specificity. Promising
candidates are the promoters of the maize genes cyclin delta 2
(Locus #GRMZM2G476685; SEQ ID NO: 22), phospholipase 2A (Locus
#GRMZM2G154523; SEQ ID NO: 23), sucrose transporter (Locus
#GRMZM2G081589; SEQ ID NO: 24), and cell wall invertase (Locus
#GRMZM2G139300; SEQ ID NO: 25) which have been shown to be
expressed in leaves but not in the fertilized ovaries at the onset
of seed development (Kakumanu et al., 2012, Plant Physiol. 160:
846-847).
Since the genes characterized in the presented invention are global
transcriptional regulators, trait modifications can also be
achieved through modulating the expression of downstream
transcription factors. For example, 10 bZIP transcription factors
regulated by the TFs of the invention were identified in transgenic
switchgrass by gene expression microarray analysis (see Example 7).
Members of the bZIP TF family have been characterized in different
plant species and linked to various developmental and physiological
processes, such as panicle and seed development, endosperm-specific
expression of storage protein genes, vegetative growth and abiotic
stress tolerance (reviewed in Nijhawan et al., 2008, Plant Physiol.
146: 333-350). In total, 18 MYB transcription factors regulated by
PvSTR1, PvSTIF1 and PvBMY1 were also identified in this study
(TABLE 8). Some of these genes are well known for their role in
major biological processes--development and cell differentiation,
photosynthesis and secondary metabolism, stress tolerance and
defense response (reviewed in Ambawat et al., 2013, Physiol. Mol.
Biol. Plants 19: 307-321) and can be useful in different approaches
to crop improvement.
Example 9
Transformation of Other Crop Species
Agrobacterium-Mediated Transformation of Miscanthus Species
Miscanthus has been extensively evaluated as a bioenergy crop in
Europe since the early 1980s (Lewandowski et al., 2003, Biomass and
Bioenergy, 25: 335-361) and, more recently, in North America
(Heaton et al., 2008, Global Change Biology, 14: 2000-2014). The
research on biomass productivity and environmental impact has
mainly been focused on M. sacchariflorus and Miscanthus x
giganteus, a pollen sterile hybrid between M. sacchariflorus and M.
sinensis (Jorgensen & Muhs, 2001, In M. B. Jones and M. Walsh
(eds.), Miscanthus for energy and fibre. James & James (Science
Publishers) Ltd., London, pp. 68-852).
For the development of tissue culture and transformation systems,
Miscanthus x giganteus plants established in soil from rhizomes and
grown under greenhouse conditions at 27.degree. C. with a 16-hour
photoperiod using supplemental sodium halide lamps (200
mol/m.sup.2/s) were used as an explant source. Immature
inflorescences, axillary meristems, and basal portions of leaves
were harvested and used for culture initiation after surface
sterilization. The initial explants and resultant cultures were
incubated at 27.degree. C., in the dark. Their response to various
concentrations and combinations of plant growth regulators and
different nitrate-to-ammonium ratios in the tissue culture medium
was tested. After 3-4 weeks of culture, the number of explants
forming callus was scored and the callus type was determined
according to visual appearance and morphogenetic ability. Callus
formation was observed from all types of explants with significant
differences in the callus induction frequency and the ratio of the
callus types formed. The results revealed that immature
inflorescences were the best explants for callus initiation and
that MS basal medium supplemented with the synthetic auxin 2,4-D as
a sole plant growth regulator was optimal for callus initiation,
induction of somatic embryo formation and suppression of precocious
plant regeneration in these cultures.
Two approaches to improving the medium for callus initiation and
growth were used. The experiments were performed with callus
cultures propagated by monthly transfers on to MS medium containing
5 mg/L of 2,4-D and 30 g/L sucrose for 6-9 months. To determine the
optimal auxin concentration for callus growth, pre-weighed pieces
of embryogenic callus (30 pieces per replication, 2 replications
per variant) were plated on MS medium supplemented with 1, 2, 3, 4,
and 5 mg/L of 2,4-D. Cultures grown on MS medium without any plant
growth regulators served as a control. After 4 weeks, all calluses
were weighed and their growth rate was calculated as %=[(callus
final fresh weight-callus initial fresh weight)/callus initial
fresh weight].times.100. Since the highest growth rate was detected
in the presence of 2 mg/L of 2,4-D, this concentration was used for
callus initiation, propagation and selection in the transformation
experiments.
For further optimization of the tissue culture procedure, the
effects of several anti-necrotic compounds on callus growth and
embryogenic response were evaluated. Briefly, pre-weighed
embryogenic callus (27-77 mg fresh weight per replication, 2
replications per variant) was plated on MS medium containing 2 mg/L
2,4-D and supplemented with ascorbic acid (15 mg/L), cysteine (40
mg/L), and silver nitrate (5 mg/L) alone or in different
combinations. Culture growth and development were monitored on a
weekly basis and callus growth rate was calculated as described
above after 4 weeks. The results showed that callus growth was
promoted by ascorbic acid and cysteine and not affected by silver
nitrate. Although the highest growth rate was detected in calluses
grown in the presence of all three anti-necrotic compounds, some
undesired changes in the development of these cultures were also
observed. Taken together, the results demonstrated that MS medium
supplemented with 2 mg/L of 2,4-D, 15 mg/L of ascorbic acid and 40
mg/L of cysteine was optimal for the growth and development of
embryogenic callus cultures.
Since young, developing panicles proved to be an excellent source
of explants for callus initiation in Miscanthus x giganteus, these
studies were further extended in order to develop a novel protocol
for in vitro production of immature inflorescences and callus
initiation from them. The possibility for vegetative propagation by
node cultures was also explored. The top culm node and the nodes
below the top one of tillers prior to flowering from plants grown
under greenhouse conditions were used as explant sources. After
surface sterilization, the nodal segments were incubated in a 10%
aqueous solution of polyvinylpyrrolidone (PVP40, Sigma), split
longitudinally and plated on to MS medium containing 10 mg/L BAP
and 30 g/L sucrose. Individual spikelets from panicles formed from
the top node were plated on the optimized medium for callus
initiation described above. Resultant calluses were propagated by
transfers every 3-4 weeks on to a fresh medium and used in
transformation experiments. For plant regeneration, calluses
initiated from in vitro developed panicles were plated on
hormone-free MS medium and incubated at 27.degree. C. with a 16 h
photoperiod (cool white fluorescent bulbs, 80 .mu.mol/m.sup.2/s)
and subcultured every 3-4 weeks. Plantlets with 3-4 leaves were
transferred to larger tissue culture containers with the same
medium and grown for another 2-3 weeks prior to transfer to
soil.
Shoots produced from nodal segments below the top node were also
cultured on hormone-free MS medium for 3-4 weeks prior to transfer
to soil.
Agrobacterium-mediated transformation of established embryogenic
callus cultures initiated from in vitro developed panicles was
performed following the previously described procedure for
switchgrass transformation (Somleva, 2006, Agrobacterium Protocols
Wang K., ed., pp 65-74: Humana Press; Somleva et al., 2002, Crop
Sci. 42: 2080-2087) with the following modifications: infected
cultures were co-cultivated with Agrobacterium tumefaciens for 5-10
days prior to transfer to a medium supplemented with 3 mg/L
bialaphos for callus selection. Using the developed methods,
Miscanthus species can be engineered with the transcription factor
genes of the invention for increased production of biomass and/or
modifications of its composition for bioenergy applications.
Miscanthus sinensis callus cultures were initiated from mature
caryopses and their embryogenic potential was evaluated as
described previously for switchgrass (U.S. Pat. No. 8,487,159 to
Somleva et al.). They were transformed following the procedure for
Agrobacterium-mediated transformation of switchgrass (Somleva,
2006, Agrobacterium Protocols Wang K., ed., pp 65-74: Humana Press;
Somleva et al., 2002, Crop Sci. 42: 2080-2087).
Agrobacterium-Mediated Transformation of Maize
The binary vectors provided in the invention can be used for
Agrobacterium-mediated transformation of maize following a
previously described procedure (Frame et al., 2006, Agrobacterium
Protocols Wang K., ed., Vol. 1, pp 185-199, Humana Press).
Plant material: Plants grown in a greenhouse are used as an explant
source. Ears are harvested 9-13 d after pollination and surface
sterilized with 80% ethanol.
Explant isolation, infection and co-cultivation: Immature zygotic
embryos (1.2-2.0 mm) are aseptically dissected from individual
kernels and incubated in A. tumefaciens strain EHA101 culture
(grown in 5 ml N6 medium supplemented with 100 .mu.M acetosyringone
for stimulation of the bacterial vir genes for 2-5 h prior to
transformation) at room temperature for 5 min. The infected embryos
are transferred scutellum side up on to a co-cultivation medium (N6
agar-solidified medium containing 300 mg/l cysteine, 5 .mu.M silver
nitrate and 100 .mu.M acetosyringone) and incubated at 20.degree.
C., in the dark for 3 d. Embryos are transferred to N6 resting
medium containing 100 mg/l cefotaxime, 100 mg/l vancomycin and 5
.mu.M silver nitrate and incubated at 28.degree. C., in the dark
for 7 d.
Callus selection: All embryos are transferred on to the first
selection medium (the resting medium described above supplemented
with 1.5 mg/l bialaphos) and incubated at 28.degree. C., in the
dark for 2 weeks followed by subculture on a selection medium
containing 3 mg/l bialaphos. Proliferating pieces of callus are
propagated and maintained by subculture on the same medium every 2
weeks.
Plant regeneration and selection: Bialaphos-resistant embryogenic
callus lines are transferred on to regeneration medium I (MS basal
medium supplemented with 60 g/l sucrose, 1.5 mg/l bialaphos and 100
mg/l cefotaxime and solidified with 3 g/l Gelrite) and incubated at
25.degree. C., in the dark for 2 to 3 weeks. Mature embryos formed
during this period are transferred on to regeneration medium II
(the same as regeneration medium I with 3 mg/l bialaphos) for
germination in the light (25.degree. C., 80-100 .mu.E/m.sup.2/s
light intensity, 16/8-h photoperiod). Regenerated plants are ready
for transfer to soil within 10-14 days.
Agrobacterium-Mediated Transformation of Sorghum
The vectors provided in the invention can be used for sorghum
transformation following a previously described procedure (Zhao,
2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 233-244,
Humana Press).
Plant material: Plants grown under greenhouse, growth chamber or
field conditions are used as an explant source. Immature panicles
are harvested 9-12 d post pollination and individual kernels are
surface sterilized with 50% bleach for 30 min followed by three
washes with sterile distilled water.
Explant isolation, infection and co-cultivation: Immature zygotic
embryos (1-1.5 mm) are aseptically dissected from individual
kernels and incubated in A. tumefaciens strain LBA4404 suspension
in PHI-I liquid medium (MS basal medium supplemented with 1 g/l
casamino acids, 1.5 mg/l 2,4-D, 68.5 g/l sucrose, 36 g/l glucose
and 100 .mu.M acetosyringone) at room temperature for 5 min. The
infected embryos are transferred with embryonic axis down on to a
co-cultivation PHI-T medium (agar-solidified modified PHI-I medium
containing 2.0 mg/l 2,4-D, 20 g/l sucrose, 10 g/l glucose, 0.5 g/l
MES, 0.7 g/l proline, 10 mg/l ascorbic acid and 100 .mu.M
acetosyringone) and incubated at 25.degree. C., in the dark for 3
d. For resting, embryos are transferred to the same medium (without
acetosyringone) supplemented with 100 mg/l carbenicillin and
incubated at 28.degree. C., in the dark for 4 d.
Callus selection: Embryos are transferred on to the first selection
medium PHI-U (PHI-T medium described above supplemented with 1.5
mg/l 2,4-D, 100 mg/l carbenicillin and 5 mg/l PPT without glucose
and acetosyringone) and incubated at 28.degree. C., in the dark for
2 weeks followed by subculture on a selection medium containing 10
mg/l PPT. Proliferating pieces of callus are propagated and
maintained by subculture on the same medium every 2 weeks for the
remainder of the callus selection process of 10 weeks.
Plant regeneration and selection: Herbicide-resistant callus is
transferred on to regeneration medium I (PHI-U medium supplemented
with 0.5 mg/l kinetin) and incubated at 28.degree. C., in the dark
for 2 to 3 weeks for callus growth and embryo development. Cultures
are transferred on to regeneration medium II (MS basal medium with
0.5 mg/l zeatin, 700 mg/l proline, 60 g/l sucrose and 100 mg/l
carbenicillin) for shoot formation (28.degree. C., in the dark).
After 2-3 weeks, shoots are transferred on to a rooting medium
(regeneration II medium supplemented with 20 g/l sucrose, 0.5 mg/l
NAA and 0.5 mg/l IBA) and grown at 25.degree. C., 270
.mu.E/m.sup.2/s light intensity with a 16/8-h photoperiod. When the
regenerated plants are 8-10 cm tall, they can be transferred to
soil and grown under greenhouse conditions.
Agrobacterium-Mediated Transformation of Barley
The vectors provided in the invention can be used for
transformation of barley as described by Tingay et al., 1997, Plant
J. 11: 1369-1376.
Plant material: Plants of the spring cultivar Golden Promise are
grown under greenhouse or growth chamber conditions at 18.degree.
C. with a 16/8 hours photoperiod. Spikes are harvested when the
zygotic embryos are 1.5-2.5 mm in length. Developing caryopses are
sterilized with sodium hypochlorite (15 w/v chlorine) for 10 min
and rinsed four times with sterile water.
Explant Isolation, Infection and Co-Cultivation:
Immature zygotic embryos are aseptically dissected from individual
kernels and after removal of the embryonic axes are placed
scutellum side up on a callus induction medium (Gelrite-solidified
MS basal medium containing 30 g/l maltose, 1.0 g/l casein
hydrolysate, 0.69 g/l proline and 2.5 mg/L dicamba. Embryos are
incubated at 24.degree. C. in the dark during subsequent culture.
One day after isolation, the embryos are incubated in A.
tumefaciens strain AGL1 culture (grown from a single colony in MG/L
medium) followed by a transfer on to the medium described
above.
Callus Selection:
After co-cultivation for 2-3 d, embryos are transferred on to the
callus induction medium supplemented with 3 mg/l bialaphos and 150
mg/l Timentin. Cultures are selected for about 2 months with
transfers to a fresh selection medium every 2 weeks.
Plant Regeneration and Selection:
Bialaphos-resistant embryogenic callus lines are transferred to a
Phytagel-solidified regeneration medium containing 1 mg/l BA and 3
mg/l bialaphos for selection of transgenic plants and grown at
24.degree. C. under fluorescent lights with a 16/8 h photoperiod.
For root development, regenerated plants are transferred to a
hormone-free callus induction medium supplemented with 1 mg/l
bialaphos. After development of a root system, plants are
transferred to soil and grown in a greenhouse or a growth chamber
under the conditions described above.
Agrobacterium-Mediated Transformation of Rice
The binary vectors provided in the invention can be used for
Agrobacterium-mediated transformation of rice following a
previously described procedure (Herve and Kayano, 2006,
Agrobacterium Protocols Wang K., ed., Vol. 1, pp 213-222, Humana
Press).
Plant material: Mature seeds from japonica rice varieties grown in
a greenhouse are used as an explant source.
Culture transformation and selection: Dehusked seeds are surface
sterilized with 70% ethanol for 1 min and 3% sodium hypochlorite
for 30 min followed by six washes with sterile distilled water.
Seeds are plated embryo side up on an induction medium
(Gelrite-solidified N6 basal medium supplemented with 300 mg/l
casamino acids, 2.88 g/l proline, 30 g/l sucrose and 2 mg/l 2,4-D)
and incubated at 32.degree. C., under continuous light for 5 d.
Germinated seeds with swelling of the scutellum are infected with
A. tumefaciens strain LBA4404 (culture from 3-day-old plates
resuspended in N6 medium supplemented with 100 .mu.M
acetosyringone, 68.5 g/l sucrose and 36 g/l glucose) at room
temperature for 2 min followed by transfer on to a co-cultivation
medium (N6 Gelrite-solidified medium containing 300 mg/l casamino
acids, 30 g/l sucrose, 10 g/l glucose, 2 mg/l 2,4-D and 100 .mu.M
acetosyringone) and incubation at 25.degree. C., in the dark for 3
d.
For selection of transformed embryogenic tissues, whole seedlings
washed with 250 mg/l cephotaxine are transferred on to N6
agar-solidified medium containing 300 mg/l casamino acids, 2.88 g/l
proline, 30 g/l sucrose, 2 mg/l 2,4-D, 100 mg/l cefotaxime, 100
mg/l vancomycin and 35 mg/l G418 disulfate). Cultures are incubated
at 32.degree. C., under continuous light for 2-3 weeks.
Plant regeneration and selection: Resistant proliferating calluses
are transferred on to agar-solidified N6 medium containing 300 mg/l
casamino acids, 500 mg/l proline, 30 g/l sucrose, 1 mg/l NAA, 5
mg/l ABA, 2 mg/l kinetin, 100 mg/l cefotaxime, 100 mg/l vancomycin
and 20 mg/l G418 disulfate. After one week of growth at 32.degree.
C., under continuous light, the surviving calluses are transferred
on to MS medium (solidified with 10 g/l agarose) supplemented with
2 g/l casamino acids, 30 g/l sucrose, 30 g/l sorbitol, 0.02 mg/l
NAA, 2 mg/l kinetin, 100 mg/l cefotaxime, 100 mg/l vancomycin and
20 mg/l G418 disulfate and incubated under the same conditions for
another week followed by a transfer on to the same medium with 7
g/l agarose. After 2 weeks, the emerging shoots are transferred on
to Gelrite-solidified MS hormone-free medium containing 30 g/l
sucrose and grown under continuous light for 1-2 weeks to promote
shoot and root development. When the regenerated plants are 8-10 cm
tall, they can be transferred to soil and grown under greenhouse
conditions. After about 10-16 weeks, transgenic seeds are
harvested.
Indica rice varieties are transformed with Agrobacterium following
a similar procedure (Datta and Datta, 2006, Agrobacterium Protocols
Wang K., ed., Vol. 1, pp 201-212, Humana Press).
Microprojectile Bombardment-Mediated Transformation of
Sugarcane
An expression cassette containing a transcription factor gene can
be co-introduced with a cassette of a marker gene (e.g., npt) into
sugarcane via biolistics following a previously described protocol
(Taparia et al., 2012, In Vitro Cell. Dev. Biol. 48: 15-22))
Plant material: Greenhouse-grown plants with 6-8 visible nodes are
used as an explant source. Tops are collected and surface
sterilized with 70% ethanol. The outermost leaves are removed under
aseptic conditions and immature leaf whorl cross sections (about 2
mm) are cut from the region 1-10 cm above the apical node.
Culture initiation, transformation and selection: The isolated leaf
sections are cultured on MS basal media supplemented with 20 g/l
sucrose, 1.86 mg/l p-chlorophenoxyacetic acid (CPA), 1.86 mg/l NAA
and 0.09 mg/l BA at 28.degree. C., under 30 .mu.mol/m.sup.2/s light
intensity and a 16/8-h photoperiod for 7 d. Embryogenic cultures
are subcultured to fresh medium and used for transformation.
For microprojectile bombardment, leaf disks are plated on the
culture initiation medium supplemented with 0.4 M sorbitol 4 hours
before gene transfer. Plasmid DNA (200 ng) containing the
expression cassettes of a TF and a marker gene is precipitated onto
1.8 mg gold particles (0.6 .mu.m) following a previously described
procedure (Altpeter and Sandhu, 2010, Genetic
transformation--biolistics, Davey & Anthony eds., pp 217-237,
Wiley, Hoboken). The DNA (10 ng per shot) is delivered to the
explants by a PDS-1000 Biolistc particle delivery system (Biorad)
using 1100-psi rupture disk, 26.5 mmHg chamber vacuum and a shelf
distance of 6 cm. pressure). The bombarded explants are transferred
to the culture initiation medium described above and incubated for
4 days.
For selection, cultures are transferred on to the initiation medium
supplemented with 30 mg/l geneticin and incubated for 10 d followed
by another selection cycle under the same conditions.
Plant regeneration and selection: Cultures are transferred on to
the selection medium described above without CPA and grown at
28.degree. C., under 100 .mu.mol/m.sup.2/s light intensity with a
16/8-h photoperiod. Leaf disks with small shoots (about 0.5 cm) are
plated on a hormone-free medium with 30 mg/l geneticin for shoot
growth and root development. Prior to transfer to soil, roots of
regenerated plants can be dipped into a commercially available root
promoting powder.
Transformation of Wheat by Microprojectile Bombardment
The gene constructs provided in the invention can be used for wheat
transformation by microprojectile bombardment following a
previously described protocol (Weeks et al., 1993, Plant Physiol.
102: 1077-1084).
Plant material: Plants from the spring wheat cultivar Bobwhite are
grown at 18-20.degree. C. day and 14-16.degree. C. night
temperatures under a 16 h photoperiod. Spikes are collected 10-12
weeks after sowing (12-16 days post anthesis). Individual caryopses
at the early-medium milk stage are sterilized with 70% ethanol for
5 min and 20% sodium hypochlorite for 15 min followed by three
washes with sterile water.
Culture initiation, transformation and selection: Immature zygotic
embryos (0.5-1.5 mm) are dissected under aseptic conditions, placed
scutellum side up on a culture induction medium
(Phytagel-solidified MS medium containing 20 g/l sucrose and 1.5
mg/l 2,4-D) and incubated at 27.degree. C., in the light (43
.mu.mol/m.sup.2/s) for 3-5 d.
For microprojectile bombardment, embryo-derived calluses are plated
on the culture initiation medium supplemented with 0.4 M sorbitol 4
hours before gene transfer. Plasmid DNA containing the expression
cassettes of a TF and the marker gene bar is precipitated onto
0.6-.mu.m gold particles and delivered to the explants as described
for sugarcane.
The bombarded explants are transferred to callus selection medium
(the culture initiation medium described above containing 1-2 mg/l
bialaphos) and subcultured every 2 weeks.
Plant regeneration and selection: After one-two selection cycles,
cultures are transferred on to MS regeneration medium supplemented
with 0.5 mg/l dicamba and 2 mg/l bialaphos. For root formation, the
resulting bialaphos-resistant shoots are transferred to
hormone-free half-strength MS medium. Plants with well-developed
roots are transferred to soil and acclimated to lower humidity at
21.degree. C. with a 16-h photoperiod (300 .mu.mol/m.sup.2/s) for
about 2 weeks prior to transfer to a greenhouse.
Agrobacterium-Mediated Transformation of Camelina
The gene constructs provided in the invention can be used for
camelina transformation by floral dip following a previously
described protocol (International Patent Application WO
2011034946).
Plant material: Plants grown from seeds under greenhouse conditions
(24.degree. C./18.degree. C. day/night temperatures) with unopened
flower buds are used for floral dip transformation.
Agrobacterium culture preparation and plant inoculation: The
constructs of interest are introduced into Agrobacterium strain
GV3101 by electroporation. A single colony of GV3101 is obtained
from a freshly streaked plate and is inoculated into 5 mL LB
medium. After overnight growth at 28.degree. C., 2 ml of culture is
transferred to a 500-mL flask containing 300 ml of LB and incubated
overnight at 28.degree. C. Cells are pelleted by centrifugation
(6000 rpm, 20 min) and diluted to an OD.sub.600 0.8 with the
infiltration medium containing 5% sucrose and 0.05% (v/v)
Silwet-L77 (Lehle Seeds, Round Rock, Tex., USA). Camelina plants
are transformed as follows. Pots containing plants at the flowering
stage are placed in a vacuum desiccator (Bel-Art, Pequannock, N.J.,
USA) and their inflorescences are immersed into the Agrobacterium
culture. A vacuum (85 kPa) is applied for 5 min. Plants are removed
from the desiccators, covered with plastic bags and kept at room
temperature, in the dark for 24 h. Plants are grown in a greenhouse
for seed formation.
Identification of transgenic seeds: To identify bialaphos-resistant
seeds, seeds from inoculated plants are harvested, sterilized with
70% ethanol and 10% bleach followed by washes with sterile water.
Sterilized seeds are placed on germination and selection medium
(half-strength MS basal medium) containing 10 mg/L bialaphos and
incubated in a growth chamber at 23/20.degree. C. (day/night) with
a 16-h photoperiod (3000 lux). Seedlings with green cotyledons are
transferred to soil about six days after initiation of
germination.
Agrobacterium-Mediated Transformation of Brassica napus
Plant material: Mature seeds are surface sterilized in 10%
commercial bleach for 30 min with gentle shaking and washed three
times with sterile distilled water.
Culture initiation and transformation: Seeds are plated on
germination medium (MS basal medium supplemented with 30 g/l
sucrose) and incubated at 24.degree. C. with a 16-h photoperiod at
a light intensity of 60-80 .mu.E/m.sup.2/s for 4-5 d. For
transformation, cotyledons with .about.2 mm of the petiole at the
base are excised from the resulting seedlings, immersed in
Agrobacterium tumefaciens strain EHA101 suspension (grown from a
single colony in 5 ml of minimal medium supplemented with
appropriate antibiotics at 28.degree. C. for 48 h) for 1 s and
immediately embedded to a depth of .about.2 mm in a co-cultivation
medium (MS basal medium with 30 g/l sucrose and 20 .mu.M
benzyladenine). The inoculated cotyledons are incubated under the
same growth conditions for 48 h.
Plant regeneration and selection: After co-cultivation, cotyledons
are transferred on to a regeneration medium comprising MS medium
supplemented with 30 g/l sucrose and 20 .mu.M benzyladenine, 300
mg/l timentin and 20 mg/l kanamycin sulfate. After 2-3 weeks,
regenerated shoots are cut and maintained on MS medium for shoot
elongation containing 30 g/l sucrose, 300 mg/l timentin, and 20
mg/l kanamycin sulfate. The elongated shoots are transferred to a
rooting medium comprising MS basal medium supplemented with 30 g/l
sucrose, 2 mg/l indole butyric acid (IBA) and 500 mg/L
carbenicillin. After root formation, plants are transferred to soil
and grown to seed maturity under growth chamber or greenhouse
conditions.
Agrobacterium-Mediated Transformation of Soybean
The soybean orthologs of the switchgrass transcription factor genes
identified in the invention (FIG. 4) are assembled in binary
vectors (TABLE 9) and used for Agrobacterium-mediated
transformation of soybean following a previously described
procedure (Ko et al., 2006, Agrobacterium Protocols Wang K., ed.,
Vol. 1, pp 397-405, Humana Press).
Plant material: Immature seeds from soybean plants grown under
greenhouse or field conditions are used as an explant source. Young
pods are harvested and surface sterilized with 70% 2-propanol for
30 sec and 25% Clorox for 20 min followed by three washes with
sterile distilled water.
Culture transformation and selection: Under aseptic conditions,
immature seeds are removed from the pods and the cotyledons are
separated from the seed coat followed by incubation in A.
tumefaciens culture (grown from a single colony at 28.degree. C.,
overnight) in co-cultivation medium (MS salts and B5 vitamins)
supplemented with 30 g/l sucrose, 40 mg/l 2,4-D and 40 mg/l
acetosyringone for 60 min. Infected explants are plated abaxial
side up on agar-solidified co-cultivation medium and incubated at
25.degree. C., in the dark for 4 d.
For selection of transformed tissues, cotyledons washed with 500
mg/l cephotaxine are placed abaxial side up on a medium for
induction of somatic embryo formation (Gelrite-solidified MS medium
containing 30 g/l sucrose, 40 mg/l 2,4-D, 500 mg/l cefotaxime, and
10 mg/l hygromycin) and incubated at 25.degree. C., under a 23-h
photoperiod (10-20 .mu.E/m.sup.2/s) for 2 weeks. After another two
weeks of growth under the same conditions in the presence of 25
mg/l hygromycin, the antibiotic-resistant somatic embryos are
transferred on MS medium for embryo maturation supplemented with 60
g/l maltose, 500 mg/l cefotaxime, and 10 mg/l hygromycin and grown
under the same conditions for 8 weeks with 2-week subculture
intervals.
Plant regeneration and selection: The resulting cotyledonary stage
embryos are desiccated at 25.degree. C., under a 23-h photoperiod
(60-80 .mu.E/m.sup.2/s) for 5-7 d followed by culture on MS
regeneration medium containing 30 g/l sucrose and 500 mg/l
cefotaxime for 4-6 weeks for shoot and root development. When the
plants are 5-10 cm tall, they are transferred to soil and grown in
a greenhouse after acclimatization for 7 d.
TABLE-US-00009 TABLE 9 Plant transformation vectors for
overexpression of the orthologous transcription factor genes
GmSTR1, GmSTIF1 and GmBMY1 in soybean. Vector ID* TF gene/marker
Annotation SEQ ID Coordinates (bp) pMBXS884 GmSTR1/hpt
Agrobacterium T-DNA right border 26 12780-12805 CaMV35S promoter to
drive GmSTR1gene 9566-11260 GmSTR1coding region 11269-12124 nos
terminator 12258-12532 CaMV35S promoter to drive hptII gene
7707-9292 hptII coding region 6456-7692 CaMV35S polyA terminator
6248-6450 Agrobacterium T-DNA left border 6173-6198 pMBXS885
GmSTIF1/hpt Agrobacterium T-DNA right border 27 12384-12409 CaMV35S
promoter to drive GmSTIF1gene 9566-11260 GmSTIF1 coding region
11269-12055 nos terminator 11862-12136 CaMV35S promoter to drive
hptII gene 7707-9292 hptII coding region 6456-7692 CaMV35S polyA
terminator 6248-6450 Agrobacterium T-DNA left border 6173-6198
pMBXS886 GmBMY1/hpt Agrobacterium T-DNA right border 28 12459-12484
CaMV35S promoter to drive GmBMY1gene 9566-11260 GmBMY1coding region
11269-11782 nos terminator 11937-12211 CaMV35S promoter to drive
hptII gene 7707-9292 hptII coding region 6456-7692 CaMV35S polyA
terminator 6248-6450 Agrobacterium T-DNA left border 6173-6198 *All
vectors are based on the transformation vector pCambia3300 found on
the world wide web at cambia.org; the hpt gene (conferring
resistance to hygromycin) is used as a marker for selection of
transformed explants and plants.
The teachings of all patents, published applications and references
cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with
references to example embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details
may be made therein without departing from the scope of the
invention encompassed by the appended claims.
SEQUENCE LISTINGS
1
771975DNAPanicum virgatum 1atgtgcggcg gggccattct cagtgatctc
tactcaccag tgaggcggac ggtcactgcc 60ggtgacctat ggggagagag tggcagcagc
aagaatgtga agaactggaa aaggagttct 120tggaagtttg atgaaggcga
tgaagacttt gaagctgatt tcaaggattt tgaggattgc 180agtagcgagg
aggaggtaga ttttggacat gaggaaaaag aattccaatt gaacagttcg
240aatttcgtgg aattcaatgg ccatactgcc aaagtcacca gcaggaagcg
aaagatccag 300taccgaggga tccggcggcg gccttggggc aaatgggcag
cagaaatcag agacccacag 360aagggcgtcc gagtttggct tggcacgttc
agcactgccg aggaagctgc aagggcatat 420gacgtggaag ctctacgcat
acgtggcaag aaagccaaga tgaatttccc taccaccatc 480acagctgctg
ggaaacacca ccggcagcgt gtggctcgac cggcaaagaa gacgtcacaa
540gagagcctga agtcaagcaa tgcctctggt catgtcatct cagcaggcag
cagtactgat 600ggcaccgttg tcaagatcga gttgtcacag tcaccagctt
ctccactacc agtgtccagc 660gcatggcttg atgcttttga gctgaagcag
cttggtggag aaacccctga agctgatggg 720agagaaaccc ctgaagaaac
tgatcatgaa acgggagtga cagcggatat gttttttggc 780aatggcgaag
tgcggctttc agatgatttt gcgtcttacg agccttaccc aaattttatg
840cagttacctt atctagaagg tgactcgtat gaaaacattg acactctttt
caacggtgaa 900gctgctcagg atggagtgaa catcggaggt ctttggaatt
tcgatgatgt gccaatggac 960cgtggtgttt actga 9752579DNAPanicum
virgatum 2atgcatatgt atcctttcta catacatgca ggttacggga cgagaatgca
ctaccgtggc 60gtgcggcggc ggccgtgggg caagtgggcg gcggagatcc gtgaccccgc
caaggcggcg 120cgtgtgtggc tcggcacctt cgacaccgcg gaggccgccg
ccgcagcgta cgacgacgcc 180gcgctccggt tcaagggcgc caaggccaag
ctcaactttc ccgagcgcgt ccgcggccgt 240accggccagg gcgcgttcct
cgtcagccct ggcgtccccc agcagccgcc gccgtcttcc 300ctgccaactg
cagccgccgc gccgacgccg ttccccggct tgatgcggta cgcgcaactc
360cagggttgga gcagcgggaa catcgcggcc agcaacaccg gtggtgatct
cgcgccgccg 420gcacaggcgt cgtcgtcggt gcagattctg gacttctcga
cgcagcaact actccggggc 480tcaccgacaa cgttcggccc accgccgacg
acgtcggcat cgatgtccag gactagcaga 540gtagatgagg cgcacgagag
ttgcgatgct cctgactga 5793654DNAPanicum virgatum 3atgccggact
ccgacaacga gtccggcggg ccgagcaacg cggagttctc gtcgccgcgg 60gagcaggacc
ggttcctgcc gatcgcgaac gtgagccgga tcatgaagaa ggcgctcccg
120gcgaacgcca agatctccaa ggacgccaag gagacggtgc aggagtgcgt
ctccgagttc 180atctccttca tcaccggcga ggcctccgac aagtgccagc
gcgagaagcg caagaccatc 240aacggcgacg acctcctctg ggccatgacc
acgctcggct tcgaggacta catcgagcca 300ctcaagctct acctccacaa
gttccgcgag ctcgagggcg agaaggtggc ctccggcgcc 360gcgggctcct
ccggctccgc ctcgcagccc cagagagaga caacgccgtc cgcgcacaat
420ggcgccgccg gggccgtcgg ctacggcatg tacggcgccg gcgccggggc
cggcggaggc 480agcggcatga tcatgatgat ggggcagccg atgtacggct
ccccaccggg cgcgtcgggg 540tacccgcagc ccccgcacca ccacatggtg
atgggcgcta aaggtggcgc ctacggccac 600ggcggcggct cgtcgccatc
gctgtcgggg ctcggcaggc aggacaggct atga 6544324PRTPanicum virgatum
4Met Cys Gly Gly Ala Ile Leu Ser Asp Leu Tyr Ser Pro Val Arg Arg1 5
10 15Thr Val Thr Ala Gly Asp Leu Trp Gly Glu Ser Gly Ser Ser Lys
Asn 20 25 30Val Lys Asn Trp Lys Arg Ser Ser Trp Lys Phe Asp Glu Gly
Asp Glu 35 40 45Asp Phe Glu Ala Asp Phe Lys Asp Phe Glu Asp Cys Ser
Ser Glu Glu 50 55 60Glu Val Asp Phe Gly His Glu Glu Lys Glu Phe Gln
Leu Asn Ser Ser65 70 75 80Asn Phe Val Glu Phe Asn Gly His Thr Ala
Lys Val Thr Ser Arg Lys 85 90 95Arg Lys Ile Gln Tyr Arg Gly Ile Arg
Arg Arg Pro Trp Gly Lys Trp 100 105 110Ala Ala Glu Ile Arg Asp Pro
Gln Lys Gly Val Arg Val Trp Leu Gly 115 120 125Thr Phe Ser Thr Ala
Glu Glu Ala Ala Arg Ala Tyr Asp Val Glu Ala 130 135 140Leu Arg Ile
Arg Gly Lys Lys Ala Lys Met Asn Phe Pro Thr Thr Ile145 150 155
160Thr Ala Ala Gly Lys His His Arg Gln Arg Val Ala Arg Pro Ala Lys
165 170 175Lys Thr Ser Gln Glu Ser Leu Lys Ser Ser Asn Ala Ser Gly
His Val 180 185 190Ile Ser Ala Gly Ser Ser Thr Asp Gly Thr Val Val
Lys Ile Glu Leu 195 200 205Ser Gln Ser Pro Ala Ser Pro Leu Pro Val
Ser Ser Ala Trp Leu Asp 210 215 220Ala Phe Glu Leu Lys Gln Leu Gly
Gly Glu Thr Pro Glu Ala Asp Gly225 230 235 240Arg Glu Thr Pro Glu
Glu Thr Asp His Glu Thr Gly Val Thr Ala Asp 245 250 255Met Phe Phe
Gly Asn Gly Glu Val Arg Leu Ser Asp Asp Phe Ala Ser 260 265 270Tyr
Glu Pro Tyr Pro Asn Phe Met Gln Leu Pro Tyr Leu Glu Gly Asp 275 280
285Ser Tyr Glu Asn Ile Asp Thr Leu Phe Asn Gly Glu Ala Ala Gln Asp
290 295 300Gly Val Asn Ile Gly Gly Leu Trp Asn Phe Asp Asp Val Pro
Met Asp305 310 315 320Arg Gly Val Tyr5192PRTPanicum virgatum 5Met
His Met Tyr Pro Phe Tyr Ile His Ala Gly Tyr Gly Thr Arg Met1 5 10
15His Tyr Arg Gly Val Arg Arg Arg Pro Trp Gly Lys Trp Ala Ala Glu
20 25 30Ile Arg Asp Pro Ala Lys Ala Ala Arg Val Trp Leu Gly Thr Phe
Asp 35 40 45Thr Ala Glu Ala Ala Ala Ala Ala Tyr Asp Asp Ala Ala Leu
Arg Phe 50 55 60Lys Gly Ala Lys Ala Lys Leu Asn Phe Pro Glu Arg Val
Arg Gly Arg65 70 75 80Thr Gly Gln Gly Ala Phe Leu Val Ser Pro Gly
Val Pro Gln Gln Pro 85 90 95Pro Pro Ser Ser Leu Pro Thr Ala Ala Ala
Ala Pro Thr Pro Phe Pro 100 105 110Gly Leu Met Arg Tyr Ala Gln Leu
Gln Gly Trp Ser Ser Gly Asn Ile 115 120 125Ala Ala Ser Asn Thr Gly
Gly Asp Leu Ala Pro Pro Ala Gln Ala Ser 130 135 140Ser Ser Val Gln
Ile Leu Asp Phe Ser Thr Gln Gln Leu Leu Arg Gly145 150 155 160Ser
Pro Thr Thr Phe Gly Pro Pro Pro Thr Thr Ser Ala Ser Met Ser 165 170
175Arg Thr Ser Arg Val Asp Glu Ala His Glu Ser Cys Asp Ala Pro Asp
180 185 1906217PRTPanicum virgatum 6Met Pro Asp Ser Asp Asn Glu Ser
Gly Gly Pro Ser Asn Ala Glu Phe1 5 10 15Ser Ser Pro Arg Glu Gln Asp
Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25 30Arg Ile Met Lys Lys Ala
Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45Ala Lys Glu Thr Val
Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60Thr Gly Glu Ala
Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile65 70 75 80Asn Gly
Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp 85 90 95Tyr
Ile Glu Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu Glu 100 105
110Gly Glu Lys Val Ala Ser Gly Ala Ala Gly Ser Ser Gly Ser Ala Ser
115 120 125Gln Pro Gln Arg Glu Thr Thr Pro Ser Ala His Asn Gly Ala
Ala Gly 130 135 140Ala Val Gly Tyr Gly Met Tyr Gly Ala Gly Ala Gly
Ala Gly Gly Gly145 150 155 160Ser Gly Met Ile Met Met Met Gly Gln
Pro Met Tyr Gly Ser Pro Pro 165 170 175Gly Ala Ser Gly Tyr Pro Gln
Pro Pro His His His Met Val Met Gly 180 185 190Ala Lys Gly Gly Ala
Tyr Gly His Gly Gly Gly Ser Ser Pro Ser Leu 195 200 205Ser Gly Leu
Gly Arg Gln Asp Arg Leu 210 2157981DNAPanicum virgatum 7atgtgcggtg
gggctattct cagtgatctc tactcaccag tgaggcggac ggtcactgcc 60ggtgacctat
ggggagagag cggcagcacc aagaatgtga agaactggaa aaggaggagt
120tcttggaagt ttgatgaaga cgatgatgac tttgaagctg atttcgagga
tttcaacgat 180tgcagtagcg aggaggaggt ggattttgta cgtgaggaaa
aagaattcca attgaacagt 240tcgaattttg tggaactcaa cggccatacc
accaaagtcg ccagcaggaa gcgaaagacc 300cagtaccgag ggatccgacg
gcgcccgtgg ggcaaatggg cagctgaaat cagagaccca 360cagaagggcg
tccgagtttg gcttggcacg ttcagcactg ccgaggaagc tgcaaaggca
420tatgacgtgg aagctctacg catacgtggc aagaaagcca aggtgaattt
ccctaacacc 480atcacagctg ctgggaaaca ccaccggcag catgtggctc
gaccagcaaa gaggatgtca 540caagagagcc tgaagtcaag cgatgcctct
ggtcatgtcg tctcagcagg cagcagtact 600gatggcaccg ttgtcaagat
tgagttgata gagtcaccag cttctccact accagtgtcc 660agcgcatggc
ttgatgcttt tgagctgaac caacttggtg gattaaggca ccttgaagct
720gatgggagag aaaccactga agaaactgat catgaaacgg gagtgacagc
agatatggtt 780tttggcgatg gcaaagtgcg gctttcagat gattttgcgt
cttacgagcc ttacccaaat 840tttatgcagt taccttacct ggaaggtaac
tcgtatgaaa acattgacac tcttttcaac 900ggtgaagccg ctcaggatgg
cgtgaacatc ggaggtctct ggaatttcga cgatgtgcca 960atggaccgtg
gtgtttacta a 98181110DNAPanicum virgatum 8atgtgcggcg gtgcgatcct
cgccaacctc accaagcagc cgggcccgcg ccggctcacg 60gagcgggacc tctggcagga
gaagaagaag cccaagaggg gcgccggcgg ggggaggcgc 120tggttcctgg
ctgaggagga tgaggacttc gaggccgact tcgaggactt ccagggcgac
180tccgatgagt cggatttgga actcggggag ggggaggacg acgacgtcgt
cgagatcaag 240cccttcgccg ccaagaggac ttcctccaaa gatggcttaa
gcaccatgac tactgctggt 300tatgatggcc ctgcagcaag gtcagccaaa
aggaagagaa agaatcaata caggggcatc 360cgccagcgcc cttggggtaa
gtgggctgct gagatcagag atcctcagaa gggtgttcgt 420gtttggcttg
gtactttcaa cagtcctgag gaagctgcaa gagcttatga tgctgaagca
480cgcaggatcc gtggtaagaa ggccaaggtt aacttccctg atgcaccaac
agttgctcag 540aagcgccgta gtgggccagc tgctgctaaa gcacccaaat
caagtgtgga acagaagcct 600accgtcaaac cagcagtgaa caaccttgcc
aacgcaaatg catcctaccc acctgctgac 660tacacctcaa gcaagccatc
tgttcagcat gccaatatgg catttcatct agcaatgaac 720tctgctagtc
ctattgagga tccagttatg aatctgcact ctgaccaggg aagtaactct
780tttgattgct cagacttgag ctgggagaat gataccaaga cttcagacat
aacatccatt 840gctcccattt ccaccatagc tgaaggtgac gagtctgcat
ttgtcaacag caatttgaac 900aactcactgg tgccttctgt tatggagaac
aatgcagttg atctcactga tgggctgaca 960gatttagaac cgtacatgag
gtttcttctg gatgatggtg caagtgagtc aattgataac 1020cttctgaacc
ttgatggatc tgaggatgtt atgagcaaca tggatctctg gagctttgat
1080gacatgcctg ctgctggcga tttctattga 111091113DNAPanicum virgatum
9atgtgcggcg gtgcgatcct cgccaacctc accaagcagc cgggcccgcg ccggctcacg
60gagcgggacc tctggcagga gaagaagaag cccaagagga gcgccggcgg gggtaggcgc
120tggttcctgg ctgaggagga tgaggacttc gaggccgact tcgaggactt
ccagggcgac 180tccgacgagt cagatttgga gctcggggag ggggaggacg
acgacgtcgt cgagatcaag 240cccttcgccg ccaagaggac ttcctccaaa
gatggcttaa gcaccatgat tactgctggt 300tatgatggcc ctgcagcaag
gtcagccaaa aggaagagaa agaatcaata caggggcatc 360cgccagcgcc
cttggggtaa gtgggctgct gagatcagag atcctcagaa gggtgttcgt
420gtctggcttg gtactttcaa cagtcctgag gaagctgcaa gagcttatga
tgctgaagca 480cgcaggatcc gtggtaagaa ggccaaggtt aacttccctg
atgcaccaac agtttctcag 540aagcgtcgta gtggcccagc tgccgctaaa
gcacccaagt taagtgtgga acagaagcct 600actgtcaaac cagcagtgaa
caaccttgcc aacgcaaatg catctttcta cccacctgct 660gactacacct
caaaccagca atttgttcag catgccaata tgccatttca tccagcaatg
720aactctgcta gtcctactga ggatccagtt atgaatctgc actctgacca
gggaagtaac 780tcttttgatt gctcagactt gagctgggag aatgatacca
agacttcaga cataacatcc 840attgctccca tttccaccat agctgaaggt
gatgagtctg catttgtcaa cagcaatttg 900aacaactcac tggtgccttc
tgttatgggg aacaatgcag ttgatctcac tgatgggctg 960acagatttag
aaccctacat gaggtttctt ctggatgatg gtgcaagtga gtcaattgat
1020aaccttctga accttgatgg atctgaggat gttatgagca acatggatct
ctggagcttt 1080gatgacatgc ctgccactgg cgatttctat tga
111310960DNAPanicum virgatum 10atgtgcgggg gcgccattct cgcggaactc
atcccgtcgc cgcgccgggc ggcgtcgaag 60ccggtgaccg cgggccacct ctggccggcg
ggctccgaca ccaagaaggc cggcagcggg 120aggagcaaga ggcaccagct
cgccgacgtc gacgactttg aggccgcctt cgaggacttc 180gccgacgatt
ttgacaagga ggaggtcgag gaccaccatt tcgtgttctc gtccaaatcc
240gcattctccc cagcccacgg cgtgcgcgcg gcgacccaga agaggcgcgg
ccgccgccac 300ttccgcggca tccggcagcg cccctggggc aagtgggcgg
cggagatccg cgacccgcac 360aagggcaccc gcgtctggct cggcaccttc
aacaccgccg aggacgccgc ccgggcctac 420gacgtcgagg cacgccgcct
ccgcggcagc aaggccaagg tcaacttccc cgcggccggc 480gcgcgcccac
gccgcggcaa cgcgccgaga ccgcagcgcc accatgccgc agcgcagccc
540gcgttgcttg caggagagaa gcggaaggag gaggagatcg tcgtgaagcc
tgaaattggg 600gcgtcgttcg acttcgacgt gggcagcttc ttcgacacgg
ccttccccgc ggcgccgccg 660gccatggaga actccttcgc cggcagcacc
gggtcggagt ccggtagccc cgcaaagaag 720atgagatacg acaacgactc
gtcgtccgat gggatgagct ccggcggcgg ctccgcgctg 780gagctcgctg
acgagctcgc gttcgatccg ttcatgctgc tccagatgcc ctactcgggc
840gggtacgagt ccctcgacgg cctgttcgcc gtcgacgccg cccaggacgt
gaacaacgac 900atgaacggcg tcagcctgtg gagcttcgac gagttccccg
acgacagcgc tgttttctaa 96011570DNAPanicum virgatum 11aacgtgacga
gaagcaggca ctaccgtggc gtgcggcggc ggccgtgggg caagtgggcg 60gcggagatcc
gtgaccccgc caaggcggcg cgcgtgtggc tcggcacctt cgacaccgcg
120gaggccgccg ctgcagcgta cgacgacgcc gcgctccggt tcaagggcgc
caaggccaag 180ctcaacttcc ccgagcgcgt ccgaggccgc accggccagg
gcgcgttcct cgtcagccct 240tgcgtccccc agcagcagcc gccgtcgccg
tcttccatgc caactgcagc cgcgccgttc 300cccggcctga tccggtatgc
acagctgctc cagggttgga acagcgggag catcgcggcc 360agcaacaccg
gtgacctcgc gccgccggcg gccttgccaa tgccgccggc acaggcgtcg
420tcgtcggtgc agattctgga cttctcgacg cagcagctcc tccggggctc
gccgacaacg 480ttcggcggcc caccgccgcc gacgtcggca tcgatgtcca
ggactagcag agtagatgag 540gcgcacgaga gttgcaatgc tcctgactga
57012558DNAPanicum virgatum 12ggtcggaggc ggcactaccg aggggtgcgg
cagcggccgt gggggaagtg ggcggcagag 60atccgggacc ccaagaaggc ggcgcgggtg
tggctgggca ccttcgacac ggcggaggac 120gccgccatcg cctacgacga
ggcggcgctc cggttcaagg gcaccaaggc caagctcaac 180ttcccggagc
gcgtccaggg ccgcaccgac ctgggcttcc tcgtcacccg cggcgtcccg
240gaccggcacc accaccaagg cgcggcggcg gcgcaggcgc agctcatgat
gctggcccgc 300ggcggcggcg gcggcgtcaa cctgccgttc ggagccgcgt
cgccgttctc gccctcgccc 360tcgccctcgt cggcgccgca gatcctggac
ttctccacgc agcagctcat ccggcccgac 420ccgccgtcgc cggccgccgc
gatgtcgtcg tcgggcgctg ctccgtccac gccgtcgtcc 480acgaccacgg
cgtcgtcgcc cggtggcggt gcatggccgt acggtgggga gcaccacagg
540aataaaaaag acgcgtga 558131089DNAPanicum virgatum 13atgtgccacg
ccgcggtggc ggactcgggg gagcagcacg ggcggcggct tctcgccgcc 60ggcgacggcg
gcggaggaga ccgccgccag cagcagcagc agccccagcc gctggagccc
120gtggtgatgg aagccaacac ggcggcgtcg ccggcgctgt cgcggggcag
gcaggcccgg 180gagatgtcgg ccatggtggc cgcgctggcc agggtggtcg
ccggctcggc gccgccggcc 240aaggcgcccc cccaggccgt gcaggatgcc
tccgcggagg aggcgtggtg gccgtacgac 300gagctcgccg ccgagccgtc
ccctgctttc gtgctcgacg gctacagcga gacgcagccg 360ctgccggagc
actactggcc ttcggctgcg gcggcgacag aggcggcgac ttcctcgcag
420acgcattacc gtgccgcctc tgctgccgcg gccgaggagg aggtaccttc
gccgtcgtcc 480gcctccgccg ccgccggggc gagcagcagc ggcagcgcgg
cgacgcggaa gcgttaccgc 540ggcgtgcggc agcgtccgtg ggggaagtgg
gcggcggaga tccgtgaccc gcacaaggcg 600gcgcgcgtgt ggctgggcac
cttcgacacc gccgaggccg ccgcccgggc ctacgatggc 660gccgcgctta
ggttccgcgg cagccgcgcc aggctcaact tccccgagtc cgccacgctc
720ccgtccccgc cgccgccgga tccggcctcg cgcgcattgc cgccgccgcc
gcccaggccg 780gacgcgcttc tggagtcgca ggctcaggcg ccctccaccg
gcggcggcat ggagcaatac 840gcggagtacg ccaggctctt gcagagcgcc
ggcggcgacc ccggcggctc atccgggacg 900ccaagtggca cgttgcctcc
ccctcctcct cctgcagcgt acagcttcgc cgcccagggc 960gtgacaccgt
tcagctacct gtcgccgccg cagagccgcg gcgagccagc aggcaacccc
1020gcggcggcgt gggcggcgag ccactaccac ggctcgtacc cgccgtggcg
gtgggaccac 1080tcaggttga 108914654DNAPanicum virgatum 14atgccggact
ccgacaaaga gtccggctgg ccgagcaacg cggagttctc gtcgccgcgg 60gagcaggacc
ggttcctgcc gatcgcgaac gtcagccgga tcatgaagat ggcgctcccg
120gcgaacgcca agatctccaa ggacgccaag gagacggtgc aggagtgcgt
ctccgagttc 180atctccttca tcaccggcga ggcctccgac aagtgccagc
gcgagaagcg caagaccatc 240aacggcgacg acctcctctg ggccatgacc
acgctcggct tcgaggacta catcgagccg 300ctcaagctct acctccacaa
gttccgcgag ctcgagggcg agaaggtggc ctccggcgcc 360gcgggctcct
ccggctccgg ctcgcagccg cagagggaga cgacgccgtc cgcgcacaat
420ggcgccggcg gggccgtcgg ctacggcatt tacggcgccg gcgccggggc
aggcggaggc 480agcggcatga tcatgatgat ggggcagccg atgtacaact
ccccaccggg cgcgtcaggg 540tacccgcagc ccccgcacca ccagatggtg
atggccgcga aaggtggcgc ctacggccac 600ggcggcggct cgtcgccgtc
gccgccgggg ctcggcaggc aggacaggct ttga 65415609DNAPanicum virgatum
15atgccggact cggacaacga ctccggcggc ccgagcaacg ccggcggcga gctgtcgtcg
60ccgcgggagc aggacaggtt cctccccatc gcgaacgtga gccggatcat gaagaaggcg
120ctcccggcga acgccaagat cagcaaggac gccaaggaga cggtgcagga
gtgcgtctcc 180gagttcatct ccttcatcac cggcgaggcc tccgacaagt
gccagcgcga gaagcgcaag 240accatcaacg gcgacgacct gctctgggcc
atgaccacgc tcggcttcga ggactacgtc 300gagccgctca agcactacct
ccacaagttc cgcgagatcg agggcgagag ggcggccgcc 360tcctcgggcg
cctcgggctc cgccgccgcg cagcagcagg gcgacgtggc gaggggcgcc
420accaatgccg gcgggtacgc cgggtacagc gccggcggca tgatgatgat
ggggcagccg 480atgtacggct cgccgcagca gcagcaccaa cagcatcaca
tggcaatggg aggcagaggc 540ggttacggcc atcaaggagg cggcggctcg
tcgtcgtcgt cggggcttgg ccggcaagac 600agggcgtga 60916543DNAPanicum
virgatum 16atggcggacg cgccagcgag ccccgggggc ggcggcggga
gccacgagag cgggagcccc 60aggggcggcg ccgggggcgg gggcggcggc gtcagggagc
aggacaggtt cctgcccatc 120gccaacatca gccgcatcat gaagaaggcc
atcccggcca acgggaagat cgccaaggac 180gccaaggaga ccgtgcagga
gtgcgtctcc gagttcatat ccttcatcac cagcgaggcg 240agtgacaagt
gccagaggga gaagaggaag accatcaacg gggacgacct actgtgggcc
300atggccacgt tggggttcga ggactacata gaacccctca aggtgtacct
gcagaagtac 360agagagatgg agggtgatag caagttaact gcaaaaactg
gcgatggctc tattaaaaag 420gatgcccttg gccatggggg agcaagtagc
tcagccacac aagggatggg ccaacaagga 480gcgtacaacc aaggaatggg
ttatatgcaa cctcagtacc ataacggaga catctcaaac 540taa
54317486DNAPanicum virgatum 17atggcggacg acggcgggag ccacgagggc
ggcggcggcg tccgggagca ggaccggttc 60ctgcccatcg ccaacatcag ccgcatcatg
aagaaggccg tcccggctaa cggcaagatc 120gccaaggatg ccaaggagac
cctgcaggag tgcgtctccg agttcatctc cttcgtcacc 180agcgaggcca
gcgacaagtg ccagaaggag aagcgcaaga ccatcaacgg cgatgatctg
240ctctgggcga tggctacgct cggattcgag gagtacgtcg agcccctcaa
gatgtaccta 300cacaagtaca gagagatgga gggtgatagt aagttgtcta
caaaggctgg tgagggctct 360gtaaagaagg atgcaattag tccccatggt
ggcaccagta gctcaagtaa ccagttggtt 420caacatggag tttacaacca
agggatgggc tatatgcaac cacagtacca taatggggat 480acctaa
48618384DNAPanicum virgatum 18atggcggacg cgccagcgag ccccgggggc
ggcggcggga gccacgagag tgggagcccc 60aagggcggcg gcgggggcgg aggcggcggc
gtcagggagc aggacaggtt cctgcccatc 120gccaacatca gccgcatcat
gaagaaggcc atcccggcca acgggaagat cgccaaggac 180gccaaggaga
ccgtgcagga gtgcgtctcc gaattcatct ccttcatcac cagcgaggcg
240agtgacaagt gccagaggga gaagaggaag accatcaacg gggacgacct
actgtgggcc 300atggccacgc tggggttcga ggactacata gaacccctca
aggtgtacct gcagaagtac 360agagaggtca caaaacactt atag
3841912168DNAArtificial SequenceSynthetic construct pMBXS809
19gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag
60gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa
120gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt
tcagtgcagc 180cgtcttctga aaacgacatg tcgcacaagt cctaagttac
gcgacaggct gccgccctgc 240ccttttcctg gcgttttctt gtcgcgtgtt
ttagtcgcat aaagtagaat acttgcgact 300agaaccggag acattacgcc
atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 360gcgtcagcac
cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct
420gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg
gagctggcca 480ggatgcttga ccacctacgc cctggcgacg ttgtgacagt
gaccaggcta gaccgcctgg 540cccgcagcac ccgcgaccta ctggacattg
ccgagcgcat ccaggaggcc ggcgcgggcc 600tgcgtagcct ggcagagccg
tgggccgaca ccaccacgcc ggccggccgc atggtgttga 660ccgtgttcgc
cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg
720ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc
ctcaccccgg 780cacagatcgc gcacgcccgc gagctgatcg accaggaagg
ccgcaccgtg aaagaggcgg 840ctgcactgct tggcgtgcat cgctcgaccc
tgtaccgcgc acttgagcgc agcgaggaag 900tgacgcccac cgaggccagg
cggcgcggtg ccttccgtga ggacgcattg accgaggccg 960acgccctggc
ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga
1020cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga
tcgcggccgg 1080gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg
ctgcatgaaa tcctggccgg 1140tttgtctgat gccaagctgg cggcctggcc
ggccagcttg gccgctgaag aaaccgagcg 1200ccgccgtcta aaaaggtgat
gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 1260gtatatgatg
cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct
1320gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct
agcccgcgcc 1380ctgcaactcg ccggggccga tgttctgtta gtcgattccg
atccccaggg cagtgcccgc 1440gattgggcgg ccgtgcggga agatcaaccg
ctaaccgttg tcggcatcga ccgcccgacg 1500attgaccgcg acgtgaaggc
catcggccgg cgcgacttcg tagtgatcga cggagcgccc 1560caggcggcgg
acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg
1620cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt
taagcagcgc 1680attgaggtca cggatggaag gctacaagcg gcctttgtcg
tgtcgcgggc gatcaaaggc 1740acgcgcatcg gcggtgaggt tgccgaggcg
ctggccgggt acgagctgcc cattcttgag 1800tcccgtatca cgcagcgcgt
gagctaccca ggcactgccg ccgccggcac aaccgttctt 1860gaatcagaac
ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa
1920tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca
aacacgctaa 1980gtgccggccg tccgagcgca cgcagcagca aggctgcaac
gttggccagc ctggcagaca 2040cgccagccat gaagcgggtc aactttcagt
tgccggcgga ggatcacacc aagctgaaga 2100tgtacgcggt acgccaaggc
aagaccatta ccgagctgct atctgaatac atcgcgcagc 2160taccagagta
aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc
2220ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat
gtgtggagga 2280acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc
ggccctgcaa tggcactgga 2340acccccaagc ccgaggaatc ggcgtgacgg
tcgcaaacca tccggcccgg tacaaatcgg 2400cgcggcgctg ggtgatgacc
tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 2460acgcatcgag
gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg
2520caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc
cgcccaaggg 2580cgacgagcaa ccagattttt tcgttccgat gctctatgac
gtgggcaccc gcgatagtcg 2640cagcatcatg gacgtggccg ttttccgtct
gtcgaagcgt gaccgacgag ctggcgaggt 2700gatccgctac gagcttccag
acgggcacgt agaggtttcc gcagggccgg ccggcatggc 2760cagtgtgtgg
gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa
2820ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac
acgttgcgga 2880cgtactcaag ttctgccggc gagccgatgg cggaaagcag
aaagacgacc tggtagaaac 2940ctgcattcgg ttaaacacca cgcacgttgc
catgcagcgt acgaagaagg ccaagaacgg 3000ccgcctggtg acggtatccg
agggtgaagc cttgattagc cgctacaaga tcgtaaagag 3060cgaaaccggg
cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat
3120cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt
tgatcgatcc 3180cggcatcggc cgttttctct accgcctggc acgccgcgcc
gcaggcaagg cagaagccag 3240atggttgttc aagacgatct acgaacgcag
tggcagcgcc ggagagttca agaagttctg 3300tttcaccgtg cgcaagctga
tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 3360ggcggggcag
gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc
3420atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag
caggggaaaa 3480aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt
gggaacccaa agccgtacat 3540tgggaaccgg aacccgtaca ttgggaaccc
aaagccgtac attgggaacc ggtcacacat 3600gtaagtgact gatataaaag
agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 3660tattaaaact
cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga
3720agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg
ccgcttcgcg 3780tcggcctatc gcggccgctg gccgctcaaa aatggctggc
ctacggccag gcaatctacc 3840agggcgcgga caagccgcgc cgtcgccact
cgaccgccgg cgcccacatc aaggcaccct 3900gcctcgcgcg tttcggtgat
gacggtgaaa acctctgaca catgcagctc ccggagacgg 3960tcacagcttg
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
4020gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
ggagtgtata 4080ctggcttaac tatgcggcat cagagcagat tgtactgaga
gtgcaccata tgcggtgtga 4140aataccgcac agatgcgtaa ggagaaaata
ccgcatcagg cgctcttccg cttcctcgct 4200cactgactcg ctgcgctcgg
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4260ggtaatacgg
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
4320ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
ataggctccg 4380cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa acccgacagg 4440actataaaga taccaggcgt ttccccctgg
aagctccctc gtgcgctctc ctgttccgac 4500cctgccgctt accggatacc
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4560tagctcacgc
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
4620gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
gtcttgagtc 4680caacccggta agacacgact tatcgccact ggcagcagcc
actggtaaca ggattagcag 4740agcgaggtat gtaggcggtg ctacagagtt
cttgaagtgg tggcctaact acggctacac 4800tagaaggaca gtatttggta
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 4860tggtagctct
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
4920gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
tttctacggg 4980gtctgacgct cagtggaacg aaaactcacg ttaagggatt
ttggtcatgc attctaggta 5040ctaaaacaat tcatccagta aaatataata
ttttattttc tcccaatcag gcttgatccc 5100cagtaagtca aaaaatagct
cgacatactg ttcttccccg atatcctccc tgatcgaccg 5160gacgcagaag
gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa
5220gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc
cgtgggaaaa 5280gacaagttcc tcttcgggct tttccgtctt taaaaaatca
tacagctcgc gcggatcttt 5340aaatggagtg tcttcttccc agttttcgca
atccacatcg gccagatcgt tattcagtaa 5400gtaatccaat tcggctaagc
ggctgtctaa gctattcgta tagggacaat ccgatatgtc 5460gatggagtga
aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg
5520ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga
gcagattgct 5580ccagccatca tgccgttcaa agtgcaggac ctttggaaca
ggcagctttc cttccagcca 5640tagcatcatg tccttttccc gttccacatc
ataggtggtc cctttatacc ggctgtccgt 5700catttttaaa tataggtttt
cattttctcc caccagctta tataccttag caggagacat 5760tccttccgta
tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat
5820tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa
gataccccaa 5880gaagctaatt ataacaagac gaactccaat tcactgttcc
ttgcattcta aaaccttaaa 5940taccagaaaa cagctttttc aaagttgttt
tcaaagttgg cgtataacat agtatcgacg 6000gagccgattt tgaaaccgcg
gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa 6060catgctaccc
tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc
6120cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc
ccgctgacgc 6180cgtcccggac tgatgggctg cctgtatcga gtggtgattt
tgtgccgagc tgccggtcgg 6240ggagctgttg gctggctggt ggcaggatat
attgtggtgt aaacaaattg acgcttagac 6300aacttaataa cacattgcgg
acgtttttaa tgtactgaat taacgccgaa ttaattcggg 6360ggatctggat
tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt
6420attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat
gagcgaaacc 6480ctataggaac cctaattccc ttatctggga actactcaca
cattattatg gagaaactcg 6540agtcaaatct cggtgacggg caggaccgga
cggggcggta ccggcaggct gaagtccagc 6600tgccagaaac ccacgtcatg
ccagttcccg tgcttgaagc cggccgcccg cagcatgccg 6660cggggggcat
atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg
6720acagcgacca cgctcttgaa gccctgtgcc tccagggact tcagcaggtg
ggtgtagagc 6780gtggagccca gtcccgtccg ctggtggcgg ggggagacgt
acacggtcga ctcggccgtc 6840cagtcgtagg cgttgcgtgc cttccagggg
cccgcgtagg cgatgccggc gacctcgccg 6900tccacctcgg cgacgagcca
gggatagcgc tcccgcagac ggacgaggtc gtccgtccac 6960tcctgcggtt
cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg
7020acgatggtgc agaccgccgg catgtccgcc tcggtggcac ggcggatgtc
ggccgggcgt 7080cgttctgggc tcatggtaga ccgcttggta tctgcattac
aatgaaatga gcaaagacta 7140tgtgagtaac actggtcaac actagggaga
aggcatcgag caagatacgt atgtaaagag 7200aagcaatata gtgtcagttg
gtagatacta gataccatca ggaggtaagg agagcaacaa 7260aaaggaaact
ctttattttt aaattttgtt acaacaaaca agcagatcaa tgcatcaaaa
7320tactgtcagt acttatttct tcagacaaca atatttaaaa caagtgcatc
tgatcttgac 7380ttatggtcac aataaaggag cagagataaa catcaaaatt
tcgtcattta tatttattcc 7440ttcaggcgtt aacaatttaa cagcacacaa
acaaaaacag aataggaata tctaattttg 7500gcaaataata agctctgcag
acgaacaaat tattatagta tcgcctataa tatgaatccc 7560tatactattg
acccatgtag tatgaagcct gtgcctaaat taacagcaaa cttctgaatc
7620caagtgccct ataacaccaa catgtgctta aataaatacc gctaagcacc
aaattacaca 7680tttctcgtat tgctgtgtag gttctatctt cgtttcgtac
taccatgtcc ctatattttg 7740ctgctacaaa ggacggcaag taatcagcac
aggcagaaca cgatttcaga gtgtaattct 7800agatccagct aaaccactct
cagcaatcac cacacaagag agcattcaga gaaacgtggc 7860agtaacaaag
gcagagggcg gagtgagcgc gtaccgaaga cggtctcgag agagatagat
7920ttgtagagag agactggtga tttcagcgtg tcctctccaa atgaaatgaa
cttccttata 7980tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca
tcccttacgt cagtggagat 8040atcacatcaa tccacttgct ttgaagacgt
ggttggaacg tcttcttttt ccacgatgct 8100cctcgtgggt gggggtccat
ctttgggacc actgtcggca gaggcatctt gaacgatagc 8160ctttccttta
tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt
8220gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata
ttaccctttg 8280ttgaaaagtc tcaatagccc tttggtcttc tgagactgta
tctttgatat tcttggagta 8340gacgagagtg tcgtgctcca ccatgttatc
acatcaatcc acttgctttg aagacgtggt 8400tggaacgtct tctttttcca
cgatgctcct cgtgggtggg ggtccatctt tgggaccact 8460gtcggcagag
gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt
8520gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc
aatggaatcc 8580gaggaggttt cccgatatta ccctttgttg aaaagtctca
atagcccttt ggtcttctga 8640gactgtatct ttgatattct tggagtagac
gagagtgtcg tgctccacca tgttggcaag 8700ctgctctagc caatacgcaa
accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 8760tggcacgaca
ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
8820tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
tatgttgtgt 8880ggaattgtga gcggataaca atttcacaca ggaaacagct
atgaccatga ttacgaattg 8940gggtttaaac cacggaagat ccaggtctcg
agactaggag acggatggga ggcgcaacgc 9000gcgatgggga ggggggcggc
gctgaccttt ctggcgaggt cgaggtagcg atcgagcagc 9060tgcagcgcgg
acacgatgag gaagacgaag atagccgcca tggacatgtt cgccagcggc
9120ggcggagcga ggctgagccg gtctctccgg cctccggtcg gcgttaagtt
ggggatcgta 9180acgtgacgtg tctcgtctcc acggatcgac acaaccggcc
tactcgggtg cacgacgccg 9240cgataagggc gagatgtccg tgcacgcagc
ccgtttggag tcctcgttgc ccacgaaccg 9300accccttaca gaacaaggcc
tagcccaaaa ctattctgag ttgagctttt gagcctagcc 9360cacctaagcc
gagcgtcatg aactgatgaa cccactacca ctagtcaagg caaaccacaa
9420ccacaaatgg atcaattgat ctagaacaat ccgaaggagg ggaggccacg
tcacactcac 9480accaaccgaa atatctgcca gaatcagatc aaccggccaa
taggacgcca gcgagcccaa 9540cacctggcga cgccgcaaaa ttcaccgcga
ggggcaccgg gcacggcaaa aacaaaagcc 9600cggcgcggtg agaatatctg
gcgactggcg gagacctggt ggccagcgcg cggccacatc 9660agccacccca
tccgcccacc tcacctccgg cgagccaatg gcaactcgtc ttaagattcc
9720acgagataag gacccgatcg ccggcgacgc tatttagcca ggtgcgcccc
ccacggtaca 9780ctccaccagc ggcatctata gcaaccggtc cagcactttc
acgctcagct tcagcaagat 9840ctaccgtctt cggtacgcgc tcactccgcc
ctctgccttt gttactgcca cgtttctctg 9900aatgctctct tgtgtggtga
ttgctgagag tggtttagct ggatctagaa ttacactctg 9960aaatcgtgtt
ctgcctgtgc tgattacttg ccgtcctttg tagcagcaaa atatagggac
10020atggtagtac gaaacgaaga tagaacctac acagcaatac gagaaatgtg
taatttggtg 10080cttagcggta tttatttaag cacatgttgg tgttataggg
cacttggatt cagaagtttg 10140ctgttaattt aggcacaggc ttcatactac
atgggtcaat agtataggga ttcatattat 10200aggcgatact ataataattt
gttcgtctgc agagcttatt atttgccaaa attagatatt 10260cctattctgt
ttttgtttgt gtgctgttaa attgttaacg cctgaaggaa taaatataaa
10320tgacgaaatt ttgatgttta tctctgctcc tttattgtga ccataagtca
agatcagatg 10380cacttgtttt aaatattgtt gtctgaagaa ataagtactg
acagtatttt gatgcattga 10440tctgcttgtt tgttgtaaca aaatttaaaa
ataaagagtt tcctttttgt tgctctcctt 10500acctcctgat ggtatctagt
atctaccaac tgatactata ttgcttctct ttacatacgt 10560atcttgctcg
atgccttctc ctagtgttga ccagtgttac tcacatagtc tttgctcatt
10620tcattgtaat gcagatacca agcggttaat taaatgtgcg gcggggccat
tctcagtgat 10680ctctactcac cagtgaggcg gacggtcact gccggtgacc
tatggggaga gagtggcagc 10740agcaagaatg tgaagaactg gaaaaggagt
tcttggaagt ttgatgaagg cgatgaagac 10800tttgaagctg atttcaagga
ttttgaggat tgcagtagcg aggaggaggt agattttgga 10860catgaggaaa
aagaattcca attgaacagt tcgaatttcg tggaattcaa tggccatact
10920gccaaagtca ccagcaggaa gcgaaagatc cagtaccgag ggatccggcg
gcggccttgg 10980ggcaaatggg cagcagaaat cagagaccca cagaagggcg
tccgagtttg gcttggcacg 11040ttcagcactg ccgaggaagc tgcaagggca
tatgacgtgg aagctctacg catacgtggc 11100aagaaagcca agatgaattt
ccctaccacc atcacagctg ctgggaaaca ccaccggcag 11160cgtgtggctc
gaccggcaaa gaagacgtca caagagagcc tgaagtcaag caatgcctct
11220ggtcatgtca tctcagcagg cagcagtact gatggcaccg ttgtcaagat
cgagttgtca 11280cagtcaccag cttctccact accagtgtcc agcgcatggc
ttgatgcttt tgagctgaag 11340cagcttggtg gagaaacccc tgaagctgat
gggagagaaa cccctgaaga aactgatcat 11400gaaacgggag tgacagcgga
tatgtttttt ggcaatggcg aagtgcggct ttcagatgat 11460tttgcgtctt
acgagcctta cccaaatttt atgcagttac cttatctaga aggtgactcg
11520tatgaaaaca ttgacactct tttcaacggt gaagctgctc aggatggagt
gaacatcgga 11580ggtctttgga atttcgatga tgtgccaatg gaccgtggtg
tttactgagg cgcgccatcg 11640ttcaaacatt tggcaataaa gtttcttaag
attgaatcct gttgccggtc ttgcgatgat 11700tatcatataa tttctgttga
attacgttaa gcatgtaata attaacatgt aatgcatgac 11760gttatttatg
agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
11820agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt
catctatgtt 11880actagatccg atgataagct gtcaaacatg acctcaggat
gaagcttggc actggccgtc 11940gttttacaac gtcgtgactg ggaaaaccct
ggcgttaccc aacttaatcg ccttgcagca 12000catccccctt tcgccagctg
gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 12060cagttgcgca
gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt
12120tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgg
121682011772DNAArtificial SequenceSynthetic construct pMBXS810
20gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag
60gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa
120gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt
tcagtgcagc 180cgtcttctga aaacgacatg tcgcacaagt cctaagttac
gcgacaggct gccgccctgc 240ccttttcctg gcgttttctt gtcgcgtgtt
ttagtcgcat aaagtagaat acttgcgact 300agaaccggag acattacgcc
atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 360gcgtcagcac
cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct
420gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg
gagctggcca 480ggatgcttga ccacctacgc cctggcgacg ttgtgacagt
gaccaggcta gaccgcctgg 540cccgcagcac ccgcgaccta ctggacattg
ccgagcgcat ccaggaggcc ggcgcgggcc 600tgcgtagcct ggcagagccg
tgggccgaca ccaccacgcc ggccggccgc atggtgttga 660ccgtgttcgc
cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg
720ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc
ctcaccccgg 780cacagatcgc gcacgcccgc gagctgatcg accaggaagg
ccgcaccgtg aaagaggcgg 840ctgcactgct tggcgtgcat cgctcgaccc
tgtaccgcgc acttgagcgc agcgaggaag 900tgacgcccac cgaggccagg
cggcgcggtg ccttccgtga ggacgcattg accgaggccg 960acgccctggc
ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga
1020cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga
tcgcggccgg 1080gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg
ctgcatgaaa tcctggccgg 1140tttgtctgat gccaagctgg cggcctggcc
ggccagcttg
gccgctgaag aaaccgagcg 1200ccgccgtcta aaaaggtgat gtgtatttga
gtaaaacagc ttgcgtcatg cggtcgctgc 1260gtatatgatg cgatgagtaa
ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 1320gtacttaacc
agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc
1380ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg
cagtgcccgc 1440gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg
tcggcatcga ccgcccgacg 1500attgaccgcg acgtgaaggc catcggccgg
cgcgacttcg tagtgatcga cggagcgccc 1560caggcggcgg acttggctgt
gtccgcgatc aaggcagccg acttcgtgct gattccggtg 1620cagccaagcc
cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc
1680attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc
gatcaaaggc 1740acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt
acgagctgcc cattcttgag 1800tcccgtatca cgcagcgcgt gagctaccca
ggcactgccg ccgccggcac aaccgttctt 1860gaatcagaac ccgagggcga
cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 1920tcaaaactca
tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa
1980gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc
ctggcagaca 2040cgccagccat gaagcgggtc aactttcagt tgccggcgga
ggatcacacc aagctgaaga 2100tgtacgcggt acgccaaggc aagaccatta
ccgagctgct atctgaatac atcgcgcagc 2160taccagagta aatgagcaaa
tgaataaatg agtagatgaa ttttagcggc taaaggaggc 2220ggcatggaaa
atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga
2280acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa
tggcactgga 2340acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca
tccggcccgg tacaaatcgg 2400cgcggcgctg ggtgatgacc tggtggagaa
gttgaaggcc gcgcaggccg cccagcggca 2460acgcatcgag gcagaagcac
gccccggtga atcgtggcaa gcggccgctg atcgaatccg 2520caaagaatcc
cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg
2580cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc
gcgatagtcg 2640cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt
gaccgacgag ctggcgaggt 2700gatccgctac gagcttccag acgggcacgt
agaggtttcc gcagggccgg ccggcatggc 2760cagtgtgtgg gattacgacc
tggtactgat ggcggtttcc catctaaccg aatccatgaa 2820ccgataccgg
gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga
2880cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc
tggtagaaac 2940ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt
acgaagaagg ccaagaacgg 3000ccgcctggtg acggtatccg agggtgaagc
cttgattagc cgctacaaga tcgtaaagag 3060cgaaaccggg cggccggagt
acatcgagat cgagctagct gattggatgt accgcgagat 3120cacagaaggc
aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc
3180cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg
cagaagccag 3240atggttgttc aagacgatct acgaacgcag tggcagcgcc
ggagagttca agaagttctg 3300tttcaccgtg cgcaagctga tcgggtcaaa
tgacctgccg gagtacgatt tgaaggagga 3360ggcggggcag gctggcccga
tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 3420atccgccggt
tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa
3480aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa
agccgtacat 3540tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac
attgggaacc ggtcacacat 3600gtaagtgact gatataaaag agaaaaaagg
cgatttttcc gcctaaaact ctttaaaact 3660tattaaaact cttaaaaccc
gcctggcctg tgcataactg tctggccagc gcacagccga 3720agagctgcaa
aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg
3780tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag
gcaatctacc 3840agggcgcgga caagccgcgc cgtcgccact cgaccgccgg
cgcccacatc aaggcaccct 3900gcctcgcgcg tttcggtgat gacggtgaaa
acctctgaca catgcagctc ccggagacgg 3960tcacagcttg tctgtaagcg
gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 4020gtgttggcgg
gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
4080ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
tgcggtgtga 4140aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
cgctcttccg cttcctcgct 4200cactgactcg ctgcgctcgg tcgttcggct
gcggcgagcg gtatcagctc actcaaaggc 4260ggtaatacgg ttatccacag
aatcagggga taacgcagga aagaacatgt gagcaaaagg 4320ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
4380cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
acccgacagg 4440actataaaga taccaggcgt ttccccctgg aagctccctc
gtgcgctctc ctgttccgac 4500cctgccgctt accggatacc tgtccgcctt
tctcccttcg ggaagcgtgg cgctttctca 4560tagctcacgc tgtaggtatc
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4620gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
4680caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag 4740agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac 4800tagaaggaca gtatttggta tctgcgctct
gctgaagcca gttaccttcg gaaaaagagt 4860tggtagctct tgatccggca
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 4920gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
4980gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc
attctaggta 5040ctaaaacaat tcatccagta aaatataata ttttattttc
tcccaatcag gcttgatccc 5100cagtaagtca aaaaatagct cgacatactg
ttcttccccg atatcctccc tgatcgaccg 5160gacgcagaag gcaatgtcat
accacttgtc cgccctgccg cttctcccaa gatcaataaa 5220gccacttact
ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa
5280gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc
gcggatcttt 5340aaatggagtg tcttcttccc agttttcgca atccacatcg
gccagatcgt tattcagtaa 5400gtaatccaat tcggctaagc ggctgtctaa
gctattcgta tagggacaat ccgatatgtc 5460gatggagtga aagagcctga
tgcactccgc atacagctcg ataatctttt cagggctttg 5520ttcatcttca
tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct
5580ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc
cttccagcca 5640tagcatcatg tccttttccc gttccacatc ataggtggtc
cctttatacc ggctgtccgt 5700catttttaaa tataggtttt cattttctcc
caccagctta tataccttag caggagacat 5760tccttccgta tcttttacgc
agcggtattt ttcgatcagt tttttcaatt ccggtgatat 5820tctcatttta
gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa
5880gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta
aaaccttaaa 5940taccagaaaa cagctttttc aaagttgttt tcaaagttgg
cgtataacat agtatcgacg 6000gagccgattt tgaaaccgcg gtgatcacag
gcagcaacgc tctgtcatcg ttacaatcaa 6060catgctaccc tccgcgagat
catccgtgtt tcaaacccgg cagcttagtt gccgttcttc 6120cgaatagcat
cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc
6180cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc
tgccggtcgg 6240ggagctgttg gctggctggt ggcaggatat attgtggtgt
aaacaaattg acgcttagac 6300aacttaataa cacattgcgg acgtttttaa
tgtactgaat taacgccgaa ttaattcggg 6360ggatctggat tttagtactg
gattttggtt ttaggaatta gaaattttat tgatagaagt 6420attttacaaa
tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc
6480ctataggaac cctaattccc ttatctggga actactcaca cattattatg
gagaaactcg 6540agtcaaatct cggtgacggg caggaccgga cggggcggta
ccggcaggct gaagtccagc 6600tgccagaaac ccacgtcatg ccagttcccg
tgcttgaagc cggccgcccg cagcatgccg 6660cggggggcat atccgagcgc
ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg 6720acagcgacca
cgctcttgaa gccctgtgcc tccagggact tcagcaggtg ggtgtagagc
6780gtggagccca gtcccgtccg ctggtggcgg ggggagacgt acacggtcga
ctcggccgtc 6840cagtcgtagg cgttgcgtgc cttccagggg cccgcgtagg
cgatgccggc gacctcgccg 6900tccacctcgg cgacgagcca gggatagcgc
tcccgcagac ggacgaggtc gtccgtccac 6960tcctgcggtt cctgcggctc
ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg 7020acgatggtgc
agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt
7080cgttctgggc tcatggtaga ccgcttggta tctgcattac aatgaaatga
gcaaagacta 7140tgtgagtaac actggtcaac actagggaga aggcatcgag
caagatacgt atgtaaagag 7200aagcaatata gtgtcagttg gtagatacta
gataccatca ggaggtaagg agagcaacaa 7260aaaggaaact ctttattttt
aaattttgtt acaacaaaca agcagatcaa tgcatcaaaa 7320tactgtcagt
acttatttct tcagacaaca atatttaaaa caagtgcatc tgatcttgac
7380ttatggtcac aataaaggag cagagataaa catcaaaatt tcgtcattta
tatttattcc 7440ttcaggcgtt aacaatttaa cagcacacaa acaaaaacag
aataggaata tctaattttg 7500gcaaataata agctctgcag acgaacaaat
tattatagta tcgcctataa tatgaatccc 7560tatactattg acccatgtag
tatgaagcct gtgcctaaat taacagcaaa cttctgaatc 7620caagtgccct
ataacaccaa catgtgctta aataaatacc gctaagcacc aaattacaca
7680tttctcgtat tgctgtgtag gttctatctt cgtttcgtac taccatgtcc
ctatattttg 7740ctgctacaaa ggacggcaag taatcagcac aggcagaaca
cgatttcaga gtgtaattct 7800agatccagct aaaccactct cagcaatcac
cacacaagag agcattcaga gaaacgtggc 7860agtaacaaag gcagagggcg
gagtgagcgc gtaccgaaga cggtctcgag agagatagat 7920ttgtagagag
agactggtga tttcagcgtg tcctctccaa atgaaatgaa cttccttata
7980tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt
cagtggagat 8040atcacatcaa tccacttgct ttgaagacgt ggttggaacg
tcttcttttt ccacgatgct 8100cctcgtgggt gggggtccat ctttgggacc
actgtcggca gaggcatctt gaacgatagc 8160ctttccttta tcgcaatgat
ggcatttgta ggtgccacct tccttttcta ctgtcctttt 8220gatgaagtga
cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg
8280ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat
tcttggagta 8340gacgagagtg tcgtgctcca ccatgttatc acatcaatcc
acttgctttg aagacgtggt 8400tggaacgtct tctttttcca cgatgctcct
cgtgggtggg ggtccatctt tgggaccact 8460gtcggcagag gcatcttgaa
cgatagcctt tcctttatcg caatgatggc atttgtaggt 8520gccaccttcc
ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc
8580gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt
ggtcttctga 8640gactgtatct ttgatattct tggagtagac gagagtgtcg
tgctccacca tgttggcaag 8700ctgctctagc caatacgcaa accgcctctc
cccgcgcgtt ggccgattca ttaatgcagc 8760tggcacgaca ggtttcccga
ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 8820tagctcactc
attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
8880ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
ttacgaattg 8940gggtttaaac cacggaagat ccaggtctcg agactaggag
acggatggga ggcgcaacgc 9000gcgatgggga ggggggcggc gctgaccttt
ctggcgaggt cgaggtagcg atcgagcagc 9060tgcagcgcgg acacgatgag
gaagacgaag atagccgcca tggacatgtt cgccagcggc 9120ggcggagcga
ggctgagccg gtctctccgg cctccggtcg gcgttaagtt ggggatcgta
9180acgtgacgtg tctcgtctcc acggatcgac acaaccggcc tactcgggtg
cacgacgccg 9240cgataagggc gagatgtccg tgcacgcagc ccgtttggag
tcctcgttgc ccacgaaccg 9300accccttaca gaacaaggcc tagcccaaaa
ctattctgag ttgagctttt gagcctagcc 9360cacctaagcc gagcgtcatg
aactgatgaa cccactacca ctagtcaagg caaaccacaa 9420ccacaaatgg
atcaattgat ctagaacaat ccgaaggagg ggaggccacg tcacactcac
9480accaaccgaa atatctgcca gaatcagatc aaccggccaa taggacgcca
gcgagcccaa 9540cacctggcga cgccgcaaaa ttcaccgcga ggggcaccgg
gcacggcaaa aacaaaagcc 9600cggcgcggtg agaatatctg gcgactggcg
gagacctggt ggccagcgcg cggccacatc 9660agccacccca tccgcccacc
tcacctccgg cgagccaatg gcaactcgtc ttaagattcc 9720acgagataag
gacccgatcg ccggcgacgc tatttagcca ggtgcgcccc ccacggtaca
9780ctccaccagc ggcatctata gcaaccggtc cagcactttc acgctcagct
tcagcaagat 9840ctaccgtctt cggtacgcgc tcactccgcc ctctgccttt
gttactgcca cgtttctctg 9900aatgctctct tgtgtggtga ttgctgagag
tggtttagct ggatctagaa ttacactctg 9960aaatcgtgtt ctgcctgtgc
tgattacttg ccgtcctttg tagcagcaaa atatagggac 10020atggtagtac
gaaacgaaga tagaacctac acagcaatac gagaaatgtg taatttggtg
10080cttagcggta tttatttaag cacatgttgg tgttataggg cacttggatt
cagaagtttg 10140ctgttaattt aggcacaggc ttcatactac atgggtcaat
agtataggga ttcatattat 10200aggcgatact ataataattt gttcgtctgc
agagcttatt atttgccaaa attagatatt 10260cctattctgt ttttgtttgt
gtgctgttaa attgttaacg cctgaaggaa taaatataaa 10320tgacgaaatt
ttgatgttta tctctgctcc tttattgtga ccataagtca agatcagatg
10380cacttgtttt aaatattgtt gtctgaagaa ataagtactg acagtatttt
gatgcattga 10440tctgcttgtt tgttgtaaca aaatttaaaa ataaagagtt
tcctttttgt tgctctcctt 10500acctcctgat ggtatctagt atctaccaac
tgatactata ttgcttctct ttacatacgt 10560atcttgctcg atgccttctc
ctagtgttga ccagtgttac tcacatagtc tttgctcatt 10620tcattgtaat
gcagatacca agcggttaat taaatgcata tgtatccttt ctacatacat
10680gcaggttacg ggacgagaat gcactaccgt ggcgtgcggc ggcggccgtg
gggcaagtgg 10740gcggcggaga tccgtgaccc cgccaaggcg gcgcgtgtgt
ggctcggcac cttcgacacc 10800gcggaggccg ccgccgcagc gtacgacgac
gccgcgctcc ggttcaaggg cgccaaggcc 10860aagctcaact ttcccgagcg
cgtccgcggc cgtaccggcc agggcgcgtt cctcgtcagc 10920cctggcgtcc
cccagcagcc gccgccgtct tccctgccaa ctgcagccgc cgcgccgacg
10980ccgttccccg gcttgatgcg gtacgcgcaa ctccagggtt ggagcagcgg
gaacatcgcg 11040gccagcaaca ccggtggtga tctcgcgccg ccggcacagg
cgtcgtcgtc ggtgcagatt 11100ctggacttct cgacgcagca actactccgg
ggctcaccga caacgttcgg cccaccgccg 11160acgacgtcgg catcgatgtc
caggactagc agagtagatg aggcgcacga gagttgcgat 11220gctcctgact
gaggcgcgcc atcgttcaaa catttggcaa taaagtttct taagattgaa
11280tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg
ttaagcatgt 11340aataattaac atgtaatgca tgacgttatt tatgagatgg
gtttttatga ttagagtccc 11400gcaattatac atttaatacg cgatagaaaa
caaaatatag cgcgcaaact aggataaatt 11460atcgcgcgcg gtgtcatcta
tgttactaga tccgatgata agctgtcaaa catgacctca 11520ggatgaagct
tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
11580acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
tagcgaagag 11640gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
atggcgaatg ctagagcagc 11700ttgagcttgg atcagattgt cgtttcccgc
cttcagttta aactatcagt gtttgacagg 11760atatattggc gg
117722112509DNAArtificial SequenceSynthetic construct pMBXS855
21gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag
60gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa
120gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt
tcagtgcagc 180cgtcttctga aaacgacatg tcgcacaagt cctaagttac
gcgacaggct gccgccctgc 240ccttttcctg gcgttttctt gtcgcgtgtt
ttagtcgcat aaagtagaat acttgcgact 300agaaccggag acattacgcc
atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 360gcgtcagcac
cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct
420gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg
gagctggcca 480ggatgcttga ccacctacgc cctggcgacg ttgtgacagt
gaccaggcta gaccgcctgg 540cccgcagcac ccgcgaccta ctggacattg
ccgagcgcat ccaggaggcc ggcgcgggcc 600tgcgtagcct ggcagagccg
tgggccgaca ccaccacgcc ggccggccgc atggtgttga 660ccgtgttcgc
cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg
720ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc
ctcaccccgg 780cacagatcgc gcacgcccgc gagctgatcg accaggaagg
ccgcaccgtg aaagaggcgg 840ctgcactgct tggcgtgcat cgctcgaccc
tgtaccgcgc acttgagcgc agcgaggaag 900tgacgcccac cgaggccagg
cggcgcggtg ccttccgtga ggacgcattg accgaggccg 960acgccctggc
ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga
1020cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga
tcgcggccgg 1080gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg
ctgcatgaaa tcctggccgg 1140tttgtctgat gccaagctgg cggcctggcc
ggccagcttg gccgctgaag aaaccgagcg 1200ccgccgtcta aaaaggtgat
gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 1260gtatatgatg
cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct
1320gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct
agcccgcgcc 1380ctgcaactcg ccggggccga tgttctgtta gtcgattccg
atccccaggg cagtgcccgc 1440gattgggcgg ccgtgcggga agatcaaccg
ctaaccgttg tcggcatcga ccgcccgacg 1500attgaccgcg acgtgaaggc
catcggccgg cgcgacttcg tagtgatcga cggagcgccc 1560caggcggcgg
acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg
1620cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt
taagcagcgc 1680attgaggtca cggatggaag gctacaagcg gcctttgtcg
tgtcgcgggc gatcaaaggc 1740acgcgcatcg gcggtgaggt tgccgaggcg
ctggccgggt acgagctgcc cattcttgag 1800tcccgtatca cgcagcgcgt
gagctaccca ggcactgccg ccgccggcac aaccgttctt 1860gaatcagaac
ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa
1920tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca
aacacgctaa 1980gtgccggccg tccgagcgca cgcagcagca aggctgcaac
gttggccagc ctggcagaca 2040cgccagccat gaagcgggtc aactttcagt
tgccggcgga ggatcacacc aagctgaaga 2100tgtacgcggt acgccaaggc
aagaccatta ccgagctgct atctgaatac atcgcgcagc 2160taccagagta
aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc
2220ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat
gtgtggagga 2280acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc
ggccctgcaa tggcactgga 2340acccccaagc ccgaggaatc ggcgtgacgg
tcgcaaacca tccggcccgg tacaaatcgg 2400cgcggcgctg ggtgatgacc
tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 2460acgcatcgag
gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg
2520caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc
cgcccaaggg 2580cgacgagcaa ccagattttt tcgttccgat gctctatgac
gtgggcaccc gcgatagtcg 2640cagcatcatg gacgtggccg ttttccgtct
gtcgaagcgt gaccgacgag ctggcgaggt 2700gatccgctac gagcttccag
acgggcacgt agaggtttcc gcagggccgg ccggcatggc 2760cagtgtgtgg
gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa
2820ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac
acgttgcgga 2880cgtactcaag ttctgccggc gagccgatgg cggaaagcag
aaagacgacc tggtagaaac 2940ctgcattcgg ttaaacacca cgcacgttgc
catgcagcgt acgaagaagg ccaagaacgg 3000ccgcctggtg acggtatccg
agggtgaagc cttgattagc cgctacaaga tcgtaaagag 3060cgaaaccggg
cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat
3120cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt
tgatcgatcc 3180cggcatcggc cgttttctct accgcctggc acgccgcgcc
gcaggcaagg cagaagccag 3240atggttgttc aagacgatct acgaacgcag
tggcagcgcc ggagagttca agaagttctg 3300tttcaccgtg cgcaagctga
tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 3360ggcggggcag
gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc
3420atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag
caggggaaaa 3480aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt
gggaacccaa agccgtacat 3540tgggaaccgg aacccgtaca ttgggaaccc
aaagccgtac attgggaacc ggtcacacat 3600gtaagtgact gatataaaag
agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 3660tattaaaact
cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga
3720agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg
ccgcttcgcg 3780tcggcctatc gcggccgctg gccgctcaaa aatggctggc
ctacggccag gcaatctacc 3840agggcgcgga caagccgcgc cgtcgccact
cgaccgccgg cgcccacatc aaggcaccct 3900gcctcgcgcg tttcggtgat
gacggtgaaa acctctgaca catgcagctc ccggagacgg 3960tcacagcttg
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
4020gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
ggagtgtata 4080ctggcttaac tatgcggcat cagagcagat tgtactgaga
gtgcaccata tgcggtgtga 4140aataccgcac agatgcgtaa ggagaaaata
ccgcatcagg cgctcttccg cttcctcgct 4200cactgactcg ctgcgctcgg
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4260ggtaatacgg
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
4320ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
gcgtttttcc ataggctccg 4380cccccctgac gagcatcaca aaaatcgacg
ctcaagtcag aggtggcgaa acccgacagg 4440actataaaga taccaggcgt
ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4500cctgccgctt
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
4560tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
tgggctgtgt 4620gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
ggtaactatc gtcttgagtc 4680caacccggta agacacgact tatcgccact
ggcagcagcc actggtaaca ggattagcag 4740agcgaggtat gtaggcggtg
ctacagagtt cttgaagtgg tggcctaact acggctacac 4800tagaaggaca
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
4860tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
ttgtttgcaa 4920gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
cctttgatct tttctacggg 4980gtctgacgct cagtggaacg aaaactcacg
ttaagggatt ttggtcatgc attctaggta 5040ctaaaacaat tcatccagta
aaatataata ttttattttc tcccaatcag gcttgatccc 5100cagtaagtca
aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg
5160gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa
gatcaataaa 5220gccacttact ttgccatctt tcacaaagat gttgctgtct
cccaggtcgc cgtgggaaaa 5280gacaagttcc tcttcgggct tttccgtctt
taaaaaatca tacagctcgc gcggatcttt 5340aaatggagtg tcttcttccc
agttttcgca atccacatcg gccagatcgt tattcagtaa 5400gtaatccaat
tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc
5460gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt
cagggctttg 5520ttcatcttca tactcttccg agcaaaggac gccatcggcc
tcactcatga gcagattgct 5580ccagccatca tgccgttcaa agtgcaggac
ctttggaaca ggcagctttc cttccagcca 5640tagcatcatg tccttttccc
gttccacatc ataggtggtc cctttatacc ggctgtccgt 5700catttttaaa
tataggtttt cattttctcc caccagctta tataccttag caggagacat
5760tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt
ccggtgatat 5820tctcatttta gccatttatt atttccttcc tcttttctac
agtatttaaa gataccccaa 5880gaagctaatt ataacaagac gaactccaat
tcactgttcc ttgcattcta aaaccttaaa 5940taccagaaaa cagctttttc
aaagttgttt tcaaagttgg cgtataacat agtatcgacg 6000gagccgattt
tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa
6060catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt
gccgttcttc 6120cgaatagcat cggtaacatg agcaaagtct gccgccttac
aacggctctc ccgctgacgc 6180cgtcccggac tgatgggctg cctgtatcga
gtggtgattt tgtgccgagc tgccggtcgg 6240ggagctgttg gctggctggt
ggcaggatat attgtggtgt aaacaaattg acgcttagac 6300aacttaataa
cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg
6360ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat
tgatagaagt 6420attttacaaa tacaaataca tactaagggt ttcttatatg
ctcaacacat gagcgaaacc 6480ctataggaac cctaattccc ttatctggga
actactcaca cattattatg gagaaactcg 6540agtcaaatct cggtgacggg
caggaccgga cggggcggta ccggcaggct gaagtccagc 6600tgccagaaac
ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg
6660cggggggcat atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg
cagcccgatg 6720acagcgacca cgctcttgaa gccctgtgcc tccagggact
tcagcaggtg ggtgtagagc 6780gtggagccca gtcccgtccg ctggtggcgg
ggggagacgt acacggtcga ctcggccgtc 6840cagtcgtagg cgttgcgtgc
cttccagggg cccgcgtagg cgatgccggc gacctcgccg 6900tccacctcgg
cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac
6960tcctgcggtt cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat
gtagtggttg 7020acgatggtgc agaccgccgg catgtccgcc tcggtggcac
ggcggatgtc ggccgggcgt 7080cgttctgggc tcatggtaga ccgcttggta
tctgcattac aatgaaatga gcaaagacta 7140tgtgagtaac actggtcaac
actagggaga aggcatcgag caagatacgt atgtaaagag 7200aagcaatata
gtgtcagttg gtagatacta gataccatca ggaggtaagg agagcaacaa
7260aaaggaaact ctttattttt aaattttgtt acaacaaaca agcagatcaa
tgcatcaaaa 7320tactgtcagt acttatttct tcagacaaca atatttaaaa
caagtgcatc tgatcttgac 7380ttatggtcac aataaaggag cagagataaa
catcaaaatt tcgtcattta tatttattcc 7440ttcaggcgtt aacaatttaa
cagcacacaa acaaaaacag aataggaata tctaattttg 7500gcaaataata
agctctgcag acgaacaaat tattatagta tcgcctataa tatgaatccc
7560tatactattg acccatgtag tatgaagcct gtgcctaaat taacagcaaa
cttctgaatc 7620caagtgccct ataacaccaa catgtgctta aataaatacc
gctaagcacc aaattacaca 7680tttctcgtat tgctgtgtag gttctatctt
cgtttcgtac taccatgtcc ctatattttg 7740ctgctacaaa ggacggcaag
taatcagcac aggcagaaca cgatttcaga gtgtaattct 7800agatccagct
aaaccactct cagcaatcac cacacaagag agcattcaga gaaacgtggc
7860agtaacaaag gcagagggcg gagtgagcgc gtaccgaaga cggtctcgag
agagatagat 7920ttgtagagag agactggtga tttcagcgtg tcctctccaa
atgaaatgaa cttccttata 7980tagaggaagg tcttgcgaag gatagtggga
ttgtgcgtca tcccttacgt cagtggagat 8040atcacatcaa tccacttgct
ttgaagacgt ggttggaacg tcttcttttt ccacgatgct 8100cctcgtgggt
gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc
8160ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta
ctgtcctttt 8220gatgaagtga cagatagctg ggcaatggaa tccgaggagg
tttcccgata ttaccctttg 8280ttgaaaagtc tcaatagccc tttggtcttc
tgagactgta tctttgatat tcttggagta 8340gacgagagtg tcgtgctcca
ccatgttatc acatcaatcc acttgctttg aagacgtggt 8400tggaacgtct
tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact
8460gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc
atttgtaggt 8520gccaccttcc ttttctactg tccttttgat gaagtgacag
atagctgggc aatggaatcc 8580gaggaggttt cccgatatta ccctttgttg
aaaagtctca atagcccttt ggtcttctga 8640gactgtatct ttgatattct
tggagtagac gagagtgtcg tgctccacca tgttggcaag 8700ctgctctagc
caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
8760tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
taatgtgagt 8820tagctcactc attaggcacc ccaggcttta cactttatgc
ttccggctcg tatgttgtgt 8880ggaattgtga gcggataaca atttcacaca
ggaaacagct atgaccatga ttacgaattg 8940gggtttaaac cacggaagat
ccaggtctcg agactaggag acggatggga ggcgcaacgc 9000gcgatgggga
ggggggcggc gctgaccttt ctggcgaggt cgaggtagcg atcgagcagc
9060tgcagcgcgg acacgatgag gaagacgaag atagccgcca tggacatgtt
cgccagcggc 9120ggcggagcga ggctgagccg gtctctccgg cctccggtcg
gcgttaagtt ggggatcgta 9180acgtgacgtg tctcgtctcc acggatcgac
acaaccggcc tactcgggtg cacgacgccg 9240cgataagggc gagatgtccg
tgcacgcagc ccgtttggag tcctcgttgc ccacgaaccg 9300accccttaca
gaacaaggcc tagcccaaaa ctattctgag ttgagctttt gagcctagcc
9360cacctaagcc gagcgtcatg aactgatgaa cccactacca ctagtcaagg
caaaccacaa 9420ccacaaatgg atcaattgat ctagaacaat ccgaaggagg
ggaggccacg tcacactcac 9480accaaccgaa atatctgcca gaatcagatc
aaccggccaa taggacgcca gcgagcccaa 9540cacctggcga cgccgcaaaa
ttcaccgcga ggggcaccgg gcacggcaaa aacaaaagcc 9600cggcgcggtg
agaatatctg gcgactggcg gagacctggt ggccagcgcg cggccacatc
9660agccacccca tccgcccacc tcacctccgg cgagccaatg gcaactcgtc
ttaagattcc 9720acgagataag gacccgatcg ccggcgacgc tatttagcca
ggtgcgcccc ccacggtaca 9780ctccaccagc ggcatctata gcaaccggtc
cagcactttc acgctcagct tcagcaagat 9840ctaccgtctt cggtacgcgc
tcactccgcc ctctgccttt gttactgcca cgtttctctg 9900aatgctctct
tgtgtggtga ttgctgagag tggtttagct ggatctagaa ttacactctg
9960aaatcgtgtt ctgcctgtgc tgattacttg ccgtcctttg tagcagcaaa
atatagggac 10020atggtagtac gaaacgaaga tagaacctac acagcaatac
gagaaatgtg taatttggtg 10080cttagcggta tttatttaag cacatgttgg
tgttataggg cacttggatt cagaagtttg 10140ctgttaattt aggcacaggc
ttcatactac atgggtcaat agtataggga ttcatattat 10200aggcgatact
ataataattt gttcgtctgc agagcttatt atttgccaaa attagatatt
10260cctattctgt ttttgtttgt gtgctgttaa attgttaacg cctgaaggaa
taaatataaa 10320tgacgaaatt ttgatgttta tctctgctcc tttattgtga
ccataagtca agatcagatg 10380cacttgtttt aaatattgtt gtctgaagaa
ataagtactg acagtatttt gatgcattga 10440tctgcttgtt tgttgtaaca
aaatttaaaa ataaagagtt tcctttttgt tgctctcctt 10500acctcctgat
ggtatctagt atctaccaac tgatactata ttgcttctct ttacatacgt
10560atcttgctcg atgccttctc ctagtgttga ccagtgttac tcacatagtc
tttgctcatt 10620tcattgtaat gcagatacca agcggttaat taaatgccgg
actccgacaa cgagtccggc 10680gggccgagca acgcggagtt ctcgtcgccg
cgggagcagg accggttcct gccgatcgcg 10740aacgtgagcc ggatcatgaa
gaaggcgctc ccggcgaacg ccaagatctc caaggacgcc 10800aaggagacgg
tgcaggagtg cgtctccgag ttcatctcct tcatcaccgg cgaggcctcc
10860gacaagtgcc agcgcgagaa gcgcaagacc atcaacggcg acgacctcct
ctgggccatg 10920accacgctcg gcttcgagga ctacatcgag ccactcaagc
tctacctcca caagttccgc 10980gagctcgagg gcgagaaggt ggcctccggc
gccgcgggct cctccggctc cgcctcgcag 11040ccccagagag agacaacgcc
gtccgcgcac aatggcgccg ccggggccgt cggctacggc 11100atgtacggcg
ccggcgccgg ggccggcgga ggcagcggca tgatcatgat gatggggcag
11160ccgatgtacg gctccccacc gggcgcgtcg gggtacccgc agcccccgca
ccaccacatg 11220gtgatgggcg ctaaaggtgg cgcctacggc cacggcggcg
gctcgtcgcc atcgctgtcg 11280gggctcggca ggcaggacag gctatgaatg
ccggactccg acaacgagtc cggcgggccg 11340agcaacgcgg agttctcgtc
gccgcgggag caggaccggt tcctgccgat cgcgaacgtg 11400agccggatca
tgaagaaggc gctcccggcg aacgccaaga tctccaagga cgccaaggag
11460acggtgcagg agtgcgtctc cgagttcatc tccttcatca ccggcgaggc
ctccgacaag 11520tgccagcgcg agaagcgcaa gaccatcaac ggcgacgacc
tcctctgggc catgaccacg 11580ctcggcttcg aggactacat cgagccactc
aagctctacc tccacaagtt ccgcgagctc 11640gagggcgaga aggtggcctc
cggcgccgcg ggctcctccg gctccgcctc gcagccccag 11700agagagacaa
cgccgtccgc gcacaatggc gccgccgggg ccgtcggcta cggcatgtac
11760ggcgccggcg ccggggccgg cggaggcagc ggcatgatca tgatgatggg
gcagccgatg 11820tacggctccc caccgggcgc gtcggggtac ccgcagcccc
cgcaccacca catggtgatg 11880ggcgctaaag gtggcgccta cggccacggc
ggcggctcgt cgccatcgct gtcggggctc 11940ggcaggcagg acaggctatg
aaactgcagg gcgcgccatc gttcaaacat ttggcaataa 12000agtttcttaa
gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg
12060aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
gagatgggtt 12120tttatgatta gagtcccgca attatacatt taatacgcga
tagaaaacaa aatatagcgc 12180gcaaactagg ataaattatc gcgcgcggtg
tcatctatgt tactagatcc gatgataagc 12240tgtcaaacat gacctcagga
tgaagcttgg cactggccgt cgttttacaa cgtcgtgact 12300gggaaaaccc
tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
12360ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
agcctgaatg 12420gcgaatgcta gagcagcttg agcttggatc agattgtcgt
ttcccgcctt cagtttaaac 12480tatcagtgtt tgacaggata tattggcgg
12509221120DNAZea mays 22cctttttacc attttctata tcctttgcat
cggcgccgta gataattgtt ggctgaaatt 60catgccagct atatgctatg tttcgaccta
ggattggctg cgcagagatg gtggtagggc 120acgccaattt atttgagata
caggttctcc atacgttcct tcacttcatt gcaatgcagc 180agagtcatat
atatacctga atcccaatcc caacaaaggt acggacctct gtgtcgtgtc
240gtcctcctcc tccggataca ttgcgtttaa tttcgaccgt atggatggat
ggatggatgt 300ggatgtggtg gccgtaatca tgtactagct tgctttgggg
ggtcatacga ttgattgatt 360gattgattgc acgggcatac caggcttcag
tgtatttgct gctctgtaga tactttactc 420atgtgaaacc cataagggtc
ggagtgagct agggcctgtg cggccggcac atagggatcg 480gacggatgga
tcggtggtgg tatgctagta tatatgcatg gtactacagc tactacccct
540cctcctcctc ctcctcccat agtgtatgtg tatgtgtatg agcagcagca
ggccgtatcg 600acaggcccaa cagacagacg atggatcaga tcggatctcc
acaccttgcc tggctcgagt 660agatcttgac catccgtgct ccaatcatgg
ccatggccgc cggactgcag agcaccaggc 720atgccatccg gaccctacta
ctactaccag tcgcttacac acctctgccc caaccgtgtc 780tcattcttgg
cagtttgggg aggaaggaag cccaatcttg tccctaaaaa acgctgttcc
840atgtaagtga ccagacgacg actatactag atcactagcc cctcgaatcc
tcgatgaaaa 900gaaaaaataa aagtcgcgag cagtcacgct cgccgaactc
aacgtccggc cgggaaggaa 960attaacggcg acagagggtc ggtccccttt
cgttcggaag tcggaactgt cattggtcgc 1020cgtcgtcgtc gcgtcactgg
catgtggggg cctcggtcgg caaaccatcg agagccgaga 1080gccgggagag
agagagagag gatggcaggt gcacatgcat 1120231020DNAZea mays 23cacatcgtgc
caagttcgag gcccattgat gcactttgct tacatatata ctcgtttaaa 60gcatgagttt
cgtgtattgt gtgtcataca cgaagcacat atatctaatt ttctctccca
120agtttcgtct aacaactaga taagataagc cttacctctt gcatgagcaa
ccaaccatac 180aaccaccacg agtgctttct cctccccctt gttgatgatg
tcgtatatta acctcaacaa 240cctaccatct ctttcctcgt ctgcttcttc
ctcacccaaa ttcttctgta ccaccataga 300tgacatcgag taggccatcc
tgctggtctc cgactcgcta accgcagcgc cccaccgcga 360caccgtcttt
accttccccc gtcgacaagc gcttcggaga gacaataagg caagaacaac
420cgagtgagag gaggagacgc tccggatctc gagtttagtt ttatgttagt
tgttgacaaa 480gaaattgtga tatattatgg tcgataataa tatatatata
ttgctgggta tcgaatgttt 540atgtgtcgtc gtaacatgcg gatatgtact
agtatatata ttatttgtca tctcaagtga 600gggacctaac catccatcac
ccgtagccaa tgacgcagtc ggatcaacga gacacaggtg 660gttgactcgg
tcggatgcgt tcgatcatgt cttagcgata gattactggt ttatcagcct
720tcgataaatg tgttgttttg agtattattc tgagtgcagg cttttgtagg
cttgtaacaa 780gtgggcagtg acaagattat taatggttgt taacaagtta
gtttcatggt gggagagtgc 840gttagcagtg tcctagatat aagcaatatc
aacttctact agttgtacag tattttattt 900ttatagatta cagtgcaaca
gtcgaccatg catctagctt tactagcggt gatcatcgtc 960gtccacgaca
caagcaatca tattctgtga cactctttcc tcgtccttat caacccaatt
102024920DNAZea mays 24acaaaagaag attagactaa tccaacagaa ttagtaaatt
cagaattctg tatggcgagt 60gaggtagact atcaaaaaag agaatgaata tgtagatgaa
gatctactaa ttttaagagc 120tatttacaaa gtctattaga gacattttct
tataataata accaaattta cctttacaaa 180ataatatgac tagtcttttg
gagttgctcc aataaaacat ataaaatggt actagtatgt 240gtgtaaacct
ttaacttctc gaaaagggac atattttttt agtgagacag aatatcatta
300gtgaaaaatt gacttttgga ttggatctga taagctaaat gggaaacgta
catgcgtcgg 360tcggtgtcca ttagttactt gacagcgtcc agctctggtc
acggtttgag attctattct 420accagagtag tgtttgaaga taagatagaa
tttaatcact atatatatat acaatcaaac 480taaacacaag tagaagtgta
atataagaag aagaaaaaaa aatctagaca atgtttggta 540tgactttaga
acaaaattct aagaaagagc tggcaagagc aataaacacc ctaactaaca
600agttgtatac tctcgcatgt aaaattgcaa ctccattaaa aacaatccaa
ttaatccaat 660ttgttgatgt tgcccctata tctttttttt tctaccaact
atactacgta tcttgatgaa 720tctccatcaa tgcttggcaa aaccccccta
ccaagaaaca gattaaggac gggaatacgg 780gatggatagc cttcccaaac
ggataaaacc ttcggcccgc cgtctcgctg ccggtggggc 840acacgccata
aaccacacgc gccggccgcc cccgcccgtg gcctttaaaa aacccccgct
900cccggcgctc gcttttcgct 92025992DNAZea mays 25agtatgccaa
ctgaaacgga tgacacatac acttcgtgaa ccaatcgata ttttacttgc 60ttctatgtta
aataatgtta taatacaata ttttattcaa atgctaaaac ttattactag
120ataaaaataa aatttaatta tcttcaaaaa ctaaccaata gatattccat
cataactaca 180tttaccaaac taatatacta aaaaatatag gataattact
aaattaatcg tgcaataatc 240agtatttatg agattgataa ttttaaattt
tgtgggctac aaacaaaaat taaaacttac 300ttttcaagtt ggagataaga
acaatggtag acgtagctcg ggatggtatg gcgtcggtgc 360agacggttac
cctttgtgcg aagtggcgcg ggcacgaggg tggggacttg gtacatgcat
420gagagagagg aagaacgaaa caacttctca aattaaagca tatgaaaatc
acctaatttt 480tgtctgtcgg tggaaactaa taactagttt ttattatctt
ttttaataag gatccacgaa 540aattattttt gaccgatgaa aatcctggat
cttcgtatta tgtttcgcct tttcccgact 600ctttgcatgc tagatttcca
tgcttggact aaaacgaaga taataaaacc aatctatcat 660tttcacacga
tgtattcata cttgcaatag ataaaccact actccgacgg gatttgcttt
720ctgacctctg aaatcttgga aggattatgt gtctacactt ctcgatcgag
gggaaaaagt 780cgtagtacca agttgtagtt aaatttgttt cttcgatgac
aaaacaaagg agaggggccc 840gcgcggcgca gcgcagcgca gttggctggt
tccggaacac gaaaaccaag cacactccac 900cagctgccat ccaccgggtt
ggatggagat tacaatactc gaatagtcag ccagccagcc 960ggcttgaacg
tgcagttttc ccctataaaa cg 9922613601DNAArtificial SequenceSynthetic
construct pMBXS884modified_base(11171)..(11176)A, T, C or
Gmisc_feature(11171)..(11176)n is a, c, g, or t 26catgccaacc
acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt
cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca
120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt
cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg
gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg
cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg
cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag
gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg
420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac
ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag
cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt
tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac
cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg
cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga
720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg
catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc
caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg
ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga
aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc
gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt
1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc
tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt
ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc
tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat
gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca
tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg
1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg
ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc
gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg
ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt
gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc
tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa
1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga
ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta
tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt
cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc
cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat
gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca
1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg
gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc
ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc
agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc
ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg
tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc
2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga
atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg
ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg
gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg
ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg
tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc
2520gatgctctat
gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg
2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc
cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat ggccagtgtg
tgggattacg acctggtact 2700gatggcggtt tcccatctaa ccgaatccat
gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc gtgttccgtc
cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820tggcggaaag
cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt
2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat
ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa gagcgaaacc
gggcggccgg agtacatcga 3000gatcgagcta gctgattgga tgtaccgcga
gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac cccgattact
ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120ggcacgccgc
gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg
3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc
tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga ggaggcgggg
caggctggcc cgatcctagt 3300catgcgctac cgcaacctga tcgagggcga
agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg caaattgccc
tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420tagcacgtac
attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa
3480cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa
aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa acttattaaa
actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc agcgcacagc
cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc tccctacgcc
ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720aaaaatggct
ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc
3780actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt
gatgacggtg 3840aaaacctctg acacatgcag ctcccggaga cggtcacagc
ttgtctgtaa gcggatgccg 3900ggagcagaca agcccgtcag ggcgcgtcag
cgggtgttgg cgggtgtcgg ggcgcagcca 3960tgacccagtc acgtagcgat
agcggagtgt atactggctt aactatgcgg catcagagca 4020gattgtactg
agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
4080ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcatagctca
cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc
tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg
caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg
attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata
4980atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata
gctcgacata 5040ctgttcttcc ccgatatcct ccctgatcga ccggacgcag
aaggcaatgt cataccactt 5100gtccgccctg ccgcttctcc caagatcaat
aaagccactt actttgccat ctttcacaaa 5160gatgttgctg tctcccaggt
cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220ctttaaaaaa
tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc
5280gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta
agcggctgtc 5340taagctattc gtatagggac aatccgatat gtcgatggag
tgaaagagcc tgatgcactc 5400cgcatacagc tcgataatct tttcagggct
ttgttcatct tcatactctt ccgagcaaag 5460gacgccatcg gcctcactca
tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520gacctttgga
acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac
5580atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt
tttcattttc 5640tcccaccagc ttatatacct tagcaggaga cattccttcc
gtatctttta cgcagcggta 5700tttttcgatc agttttttca attccggtga
tattctcatt ttagccattt attatttcct 5760tcctcttttc tacagtattt
aaagataccc caagaagcta attataacaa gacgaactcc 5820aattcactgt
tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg
5880ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc
gcggtgatca 5940caggcagcaa cgctctgtca tcgttacaat caacatgcta
ccctccgcga gatcatccgt 6000gtttcaaacc cggcagctta gttgccgttc
ttccgaatag catcggtaac atgagcaaag 6060tctgccgcct tacaacggct
ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120cgagtggtga
ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga
6180tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg
cggacgtttt 6240taatgtactg aattaacgcc gaattaattc gggggatctg
gattttagta ctggattttg 6300gttttaggaa ttagaaattt tattgataga
agtattttac aaatacaaat acatactaag 6360ggtttcttat atgctcaaca
catgagcgaa accctatagg aaccctaatt cccttatctg 6420ggaactactc
acacattatt atggagaaac tcgagggatc ccggtcggca tctactctat
6480tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag
tacttctaca 6540cagccatcgg tccagacggc cgcgcttctg cgggcgattt
gtgtacgccc gacagtcccg 6600gctccggatc ggacgattgc gtcgcatcga
ccctgcgccc aagctgcatc atcgaaattg 6660ccgtcaacca agctctgata
gagttggtca agaccaatgc ggagcatata cgcccggagc 6720cgcggcgatc
ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
6780caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga
atccccgaac 6840atcgcctcgc tccagtcaat gaccgctgtt atgcggccat
tgtccgtcag gacattgttg 6900gagccgaaat ccgcgtgcac gaggtgccgg
acttcggggc agtcctcggc ccaaagcatc 6960agctcatcga gagcctgcgc
gacggacgca ctgacggtgt cgtccatcac agtttgccag 7020tgatacacat
ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
7080attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc
cgcagcgatc 7140gcatccatgg cctccgcgac cggctgcagt tatcatcatc
atcatagaca cacgaaataa 7200agtaatcaga ttatcagtta aagctatgta
atatttacac cataaccaat caattaaaaa 7260atagatcagt ttaaagaaag
atcaaagctc aaaaaaataa aaagagaaaa gggtcctaac 7320caagaaaatg
aaggagaaaa actagaaatt tacctgcaga acagcgggca gttcggtttc
7380aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg
tcaggctctc 7440gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
gcggccgatg caaagtgccg 7500ataaacataa cgatctttgt agaaaccatc
ggcgcagcta tttacccgca ggacatatcc 7560acgccctcct acatcgaagc
tgaaagcacg agattcttcg ccctccgaga gctgcatcag 7620gtcggagacg
ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc
7680aggctttttc atggtagagg agctcgccgc ttggtatctg cattacaatg
aaatgagcaa 7740agactatgtg agtaacactg gtcaacacta gggagaaggc
atcgagcaag atacgtatgt 7800aaagagaagc aatatagtgt cagttggtag
atactagata ccatcaggag gtaaggagag 7860caacaaaaag gaaactcttt
atttttaaat tttgttacaa caaacaagca gatcaatgca 7920tcaaaatact
gtcagtactt atttcttcag acaacaatat ttaaaacaag tgcatctgat
7980cttgacttat ggtcacaata aaggagcaga gataaacatc aaaatttcgt
catttatatt 8040tattccttca ggcgttaaca atttaacagc acacaaacaa
aaacagaata ggaatatcta 8100attttggcaa ataataagct ctgcagacga
acaaattatt atagtatcgc ctataatatg 8160aatccctata ctattgaccc
atgtagtatg aagcctgtgc ctaaattaac agcaaacttc 8220tgaatccaag
tgccctataa caccaacatg tgcttaaata aataccgcta agcaccaaat
8280tacacatttc tcgtattgct gtgtaggttc tatcttcgtt tcgtactacc
atgtccctat 8340attttgctgc tacaaaggac ggcaagtaat cagcacaggc
agaacacgat ttcagagtgt 8400aattctagat ccagctaaac cactctcagc
aatcaccaca caagagagca ttcagagaaa 8460cgtggcagta acaaaggcag
agggcggagt gagcgcgtac cgaagacggt agatctctcg 8520agagagatag
atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg
8580aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt
catcccttac 8640gtcagtggag atatcacatc aatccacttg ctttgaagac
gtggttggaa cgtcttcttt 8700ttccacgatg ctcctcgtgg gtgggggtcc
atctttggga ccactgtcgg cagaggcatc 8760ttgaacgata gcctttcctt
tatcgcaatg atggcatttg taggtgccac cttccttttc 8820tactgtcctt
ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga
8880tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg
tatctttgat 8940attcttggag tagacgagag tgtcgtgctc caccatgtta
tcacatcaat ccacttgctt 9000tgaagacgtg gttggaacgt cttctttttc
cacgatgctc ctcgtgggtg ggggtccatc 9060tttgggacca ctgtcggcag
aggcatcttg aacgatagcc tttcctttat cgcaatgatg 9120gcatttgtag
gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg
9180gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct
caatagccct 9240ttggtcttct gagactgtat ctttgatatt cttggagtag
acgagagtgt cgtgctccac 9300catgttggca agctgctcta gccaatacgc
aaaccgcctc tccccgcgcg ttggccgatt 9360cattaatgca gctggcacga
caggtttccc gactggaaag cgggcagtga gcgcaacgca 9420attaatgtga
gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
9480cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
ctatgaccat 9540gattacgaat tcgagctcgg taccccacgg aagatccagg
tctcgagact aggagacgga 9600tgggaggcgc aacgcgcgat ggggaggggg
gcggcgctga cctttctggc gaggtcgagg 9660tagcgatcga gcagctgcag
cgcggacacg atgaggaaga cgaagatagc cgccatggac 9720atgttcgcca
gcggcggcgg agcgaggctg agccggtctc tccggcctcc ggtcggcgtt
9780aagttgggga tcgtaacgtg acgtgtctcg tctccacgga tcgacacaac
cggcctactc 9840gggtgcacga cgccgcgata agggcgagat gtccgtgcac
gcagcccgtt tggagtcctc 9900gttgcccacg aaccgacccc ttacagaaca
aggcctagcc caaaactatt ctgagttgag 9960cttttgagcc tagcccacct
aagccgagcg tcatgaactg atgaacccac taccactagt 10020caaggcaaac
cacaaccaca aatggatcaa ttgatctaga acaatccgaa ggaggggagg
10080ccacgtcaca ctcacaccaa ccgaaatatc tgccagaatc agatcaaccg
gccaatagga 10140cgccagcgag cccaacacct ggcgacgccg caaaattcac
cgcgaggggc accgggcacg 10200gcaaaaacaa aagcccggcg cggtgagaat
atctggcgac tggcggagac ctggtggcca 10260gcgcgcggcc acatcagcca
ccccatccgc ccacctcacc tccggcgagc caatggcaac 10320tcgtcttaag
attccacgag ataaggaccc gatcgccggc gacgctattt agccaggtgc
10380gccccccacg gtacactcca ccagcggcat ctatagcaac cggtccagca
ctttcacgct 10440cagcttcagc aagatctacc gtcttcggta cgcgctcact
ccgccctctg cctttgttac 10500tgccacgttt ctctgaatgc tctcttgtgt
ggtgattgct gagagtggtt tagctggatc 10560tagaattaca ctctgaaatc
gtgttctgcc tgtgctgatt acttgccgtc ctttgtagca 10620gcaaaatata
gggacatggt agtacgaaac gaagatagaa cctacacagc aatacgagaa
10680atgtgtaatt tggtgcttag cggtatttat ttaagcacat gttggtgtta
tagggcactt 10740ggattcagaa gtttgctgtt aatttaggca caggcttcat
actacatggg tcaatagtat 10800agggattcat attataggcg atactataat
aatttgttcg tctgcagagc ttattatttg 10860ccaaaattag atattcctat
tctgtttttg tttgtgtgct gttaaattgt taacgcctga 10920aggaataaat
ataaatgacg aaattttgat gtttatctct gctcctttat tgtgaccata
10980agtcaagatc agatgcactt gttttaaata ttgttgtctg aagaaataag
tactgacagt 11040attttgatgc attgatctgc ttgtttgttg taacaaaatt
taaaaataaa gagtttcctt 11100tttgttgctc tccttacctc ctgatggtat
ctagtatcta ccaactgata ctatattgct 11160tctctttaca nnnnnntctt
gctcgatgcc ttctcctagt gttgaccagt gttactcaca 11220tagtctttgc
tcatttcatt gtaatgcaga taccaagcgg ttaattaact atgagtcttt
11280tccttttacg attcctccac ttctccaact acatcaaagg gagtacaacc
gcaaagtccg 11340tagccttcca ggtgcgcgct gagaaattcg cgaaccgcaa
gcgtaagaat cagtatagag 11400gcatacgcca gagaccgtgg ggtaagtggg
ccgccgaaat ccgtgatcca cgtaagggag 11460tgcgagtctg gcttggcacg
ttcaatactg cagaagaagc ggcgagggcg tatgatgcag 11520aggcaaggcg
tataaggggt aagaaagcga aagttaattt tcctgaggag gctcccggga
11580cctctgtcaa acgttccaaa gtgaatcccc aggaaaacct ttcgcacaaa
ttcggcgccg 11640gcaacaatca catggatttg gtggagcaga agccgctggt
taatcagtac gcaaacatgg 11700cgtcatttcc ggggagcggg aatggattaa
cctctctacc aagtagcgat gacgtgacac 11760tatacttcag tagcgaccag
ggctccaact catttgggtg gtccgagcag gggccgaaaa 11820ctcctgaaat
aagcagcatg ttaagcgccc cactcgattg tgaatctcat ttcgtacaaa
11880atgctaacca acagccgaat tcacagaatg tcgtgtccat ggaggatgac
tcagctaaaa 11940ggctgagcga agaacgcgtt gatattgagt cggagctaaa
attcttccaa atggcgtact 12000tggaaggatc atggggcgac acaagtctcg
agtcgctcct gtcgggagat acgacgcaag 12060acggcgggaa tctaatgaat
ctatggagct tcgatgatat tccatcaatg tcttctggcg 12120tgtttatgag
tcttttcctt ttacgattcc tccacttctc caactacatc aaagggagta
12180caaccgcaaa gtccgtagcc ttccaggtgc gcgctgagaa attcgcgaac
cgcaagcgta 12240agaatcagta tagaggcata cgccagagac cgtggggtaa
gtgggccgcc gaaatccgtg 12300atccacgtaa gggagtgcga gtctggcttg
gcacgttcaa tactgcagaa gaagcggcga 12360gggcgtatga tgcagaggca
aggcgtataa ggggtaagaa agcgaaagtt aattttcctg 12420aggaggctcc
cgggacctct gtcaaacgtt ccaaagtgaa tccccaggaa aacctttcgc
12480acaaattcgg cgccggcaac aatcacatgg atttggtgga gcagaagccg
ctggttaatc 12540agtacgcaaa catggcgtca tttccgggga gcgggaatgg
attaacctct ctaccaagta 12600gcgatgacgt gacactatac ttcagtagcg
accagggctc caactcattt gggtggtccg 12660agcaggggcc gaaaactcct
gaaataagca gcatgttaag cgccccactc gattgtgaat 12720ctcatttcgt
acaaaatgct aaccaacagc cgaattcaca gaatgtcgtg tccatggagg
12780atgactcagc taaaaggctg agcgaagaac gcgttgatat tgagtcggag
ctaaaattct 12840tccaaatggc gtacttggaa ggatcatggg gcgacacaag
tctcgagtcg ctcctgtcgg 12900gagatacgac gcaagacggc gggaatctaa
tgaatctatg gagcttcgat gatattccat 12960caatgtcttc tggcgtgttt
gcagggcgcg ccatcgttca aacatttggc aataaagttt 13020cttaagattg
aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta
13080cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
gggtttttat 13140gattagagtc ccgcaattat acatttaata cgcgatagaa
aacaaaatat agcgcgcaaa 13200ctaggataaa ttatcgcgcg cggtgtcatc
tatgttacta gatccgatga taagctgtca 13260aacatgaaag cttggcactg
gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 13320ttacccaact
taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
13380aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
tgctagagca 13440gcttgagctt ggatcagatt gtcgtttccc gccttcagtt
taaactatca gtgtttgaca 13500ggatatattg gcgggtaaac ctaagagaaa
agagcgttta ttagaataac ggatatttaa 13560aagggcgtga aaaggtttat
ccgttcgtcc atttgtatgt g 136012712866DNAArtificial SequenceSynthetic
construct pMBXS885modified_base(11171)..(11176)A, T, C or
Gmisc_feature(11171)..(11176)n is a, c, g, or t 27catgccaacc
acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt
cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca
120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt
cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg
gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg
cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg
cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag
gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg
420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac
ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag
cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt
tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac
cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg
cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga
720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg
catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc
caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg
ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga
aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc
gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt
1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc
tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt
ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc
tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat
gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca
tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg
1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg
ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc
gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg
ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt
gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc
tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa
1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga
ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta
tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt
cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc
cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat
gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca
1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg
gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc
ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc
agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc
ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg
tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc
2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga
atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg
ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg
gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg
ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg
tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc
2520gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg
ccgttttccg 2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc
tacgagcttc cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat
ggccagtgtg tgggattacg acctggtact 2700gatggcggtt tcccatctaa
ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc
gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga
2820tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca
ccacgcacgt 2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg
gtgacggtat ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa
gagcgaaacc gggcggccgg agtacatcga 3000gatcgagcta gctgattgga
tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac
cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct
3120ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga
tctacgaacg 3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc
gtgcgcaagc tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga
ggaggcgggg caggctggcc cgatcctagt 3300catgcgctac cgcaacctga
tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg
caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga
3420tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt
acattgggaa 3480cccaaagccg tacattggga accggtcaca catgtaagtg
actgatataa aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa
acttattaaa actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc
agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc
tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc
3720aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg
cgccgtcgcc 3780actcgaccgc cggcgcccac
atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840aaaacctctg
acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
3900ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
ggcgcagcca 3960tgacccagtc acgtagcgat agcggagtgt atactggctt
aactatgcgg catcagagca 4020gattgtactg agagtgcacc atatgcggtg
tgaaataccg cacagatgcg taaggagaaa 4080ataccgcatc aggcgctctt
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta
actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgcattctag
gtactaaaac aattcatcca gtaaaatata 4980atattttatt ttctcccaat
caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040ctgttcttcc
ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt
5100gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat
ctttcacaaa 5160gatgttgctg tctcccaggt cgccgtggga aaagacaagt
tcctcttcgg gcttttccgt 5220ctttaaaaaa tcatacagct cgcgcggatc
tttaaatgga gtgtcttctt cccagttttc 5280gcaatccaca tcggccagat
cgttattcag taagtaatcc aattcggcta agcggctgtc 5340taagctattc
gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc
5400cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt
ccgagcaaag 5460gacgccatcg gcctcactca tgagcagatt gctccagcca
tcatgccgtt caaagtgcag 5520gacctttgga acaggcagct ttccttccag
ccatagcatc atgtcctttt cccgttccac 5580atcataggtg gtccctttat
accggctgtc cgtcattttt aaatataggt tttcattttc 5640tcccaccagc
ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta
5700tttttcgatc agttttttca attccggtga tattctcatt ttagccattt
attatttcct 5760tcctcttttc tacagtattt aaagataccc caagaagcta
attataacaa gacgaactcc 5820aattcactgt tccttgcatt ctaaaacctt
aaataccaga aaacagcttt ttcaaagttg 5880ttttcaaagt tggcgtataa
catagtatcg acggagccga ttttgaaacc gcggtgatca 5940caggcagcaa
cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt
6000gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac
atgagcaaag 6060tctgccgcct tacaacggct ctcccgctga cgccgtcccg
gactgatggg ctgcctgtat 6120cgagtggtga ttttgtgccg agctgccggt
cggggagctg ttggctggct ggtggcagga 6180tatattgtgg tgtaaacaaa
ttgacgctta gacaacttaa taacacattg cggacgtttt 6240taatgtactg
aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg
6300gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat
acatactaag 6360ggtttcttat atgctcaaca catgagcgaa accctatagg
aaccctaatt cccttatctg 6420ggaactactc acacattatt atggagaaac
tcgagggatc ccggtcggca tctactctat 6480tcctttgccc tcggacgagt
gctggggcgt cggtttccac tatcggcgag tacttctaca 6540cagccatcgg
tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg
6600gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc
atcgaaattg 6660ccgtcaacca agctctgata gagttggtca agaccaatgc
ggagcatata cgcccggagc 6720cgcggcgatc ctgcaagctc cggatgcctc
cgctcgaagt agcgcgtctg ctgctccata 6780caagccaacc acggcctcca
gaagaagatg ttggcgacct cgtattggga atccccgaac 6840atcgcctcgc
tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg
6900gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc
ccaaagcatc 6960agctcatcga gagcctgcgc gacggacgca ctgacggtgt
cgtccatcac agtttgccag 7020tgatacacat ggggatcagc aatcgcgcat
atgaaatcac gccatgtagt gtattgaccg 7080attccttgcg gtccgaatgg
gccgaacccg ctcgtctggc taagatcggc cgcagcgatc 7140gcatccatgg
cctccgcgac cggctgcagt tatcatcatc atcatagaca cacgaaataa
7200agtaatcaga ttatcagtta aagctatgta atatttacac cataaccaat
caattaaaaa 7260atagatcagt ttaaagaaag atcaaagctc aaaaaaataa
aaagagaaaa gggtcctaac 7320caagaaaatg aaggagaaaa actagaaatt
tacctgcaga acagcgggca gttcggtttc 7380aggcaggtct tgcaacgtga
caccctgtgc acggcgggag atgcaatagg tcaggctctc 7440gctgaattcc
ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
7500ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca
ggacatatcc 7560acgccctcct acatcgaagc tgaaagcacg agattcttcg
ccctccgaga gctgcatcag 7620gtcggagacg ctgtcgaact tttcgatcag
aaacttctcg acagacgtcg cggtgagttc 7680aggctttttc atggtagagg
agctcgccgc ttggtatctg cattacaatg aaatgagcaa 7740agactatgtg
agtaacactg gtcaacacta gggagaaggc atcgagcaag atacgtatgt
7800aaagagaagc aatatagtgt cagttggtag atactagata ccatcaggag
gtaaggagag 7860caacaaaaag gaaactcttt atttttaaat tttgttacaa
caaacaagca gatcaatgca 7920tcaaaatact gtcagtactt atttcttcag
acaacaatat ttaaaacaag tgcatctgat 7980cttgacttat ggtcacaata
aaggagcaga gataaacatc aaaatttcgt catttatatt 8040tattccttca
ggcgttaaca atttaacagc acacaaacaa aaacagaata ggaatatcta
8100attttggcaa ataataagct ctgcagacga acaaattatt atagtatcgc
ctataatatg 8160aatccctata ctattgaccc atgtagtatg aagcctgtgc
ctaaattaac agcaaacttc 8220tgaatccaag tgccctataa caccaacatg
tgcttaaata aataccgcta agcaccaaat 8280tacacatttc tcgtattgct
gtgtaggttc tatcttcgtt tcgtactacc atgtccctat 8340attttgctgc
tacaaaggac ggcaagtaat cagcacaggc agaacacgat ttcagagtgt
8400aattctagat ccagctaaac cactctcagc aatcaccaca caagagagca
ttcagagaaa 8460cgtggcagta acaaaggcag agggcggagt gagcgcgtac
cgaagacggt agatctctcg 8520agagagatag atttgtagag agagactggt
gatttcagcg tgtcctctcc aaatgaaatg 8580aacttcctta tatagaggaa
ggtcttgcga aggatagtgg gattgtgcgt catcccttac 8640gtcagtggag
atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt
8700ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg
cagaggcatc 8760ttgaacgata gcctttcctt tatcgcaatg atggcatttg
taggtgccac cttccttttc 8820tactgtcctt ttgatgaagt gacagatagc
tgggcaatgg aatccgagga ggtttcccga 8880tattaccctt tgttgaaaag
tctcaatagc cctttggtct tctgagactg tatctttgat 8940attcttggag
tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt
9000tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg
ggggtccatc 9060tttgggacca ctgtcggcag aggcatcttg aacgatagcc
tttcctttat cgcaatgatg 9120gcatttgtag gtgccacctt ccttttctac
tgtccttttg atgaagtgac agatagctgg 9180gcaatggaat ccgaggaggt
ttcccgatat taccctttgt tgaaaagtct caatagccct 9240ttggtcttct
gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac
9300catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg
ttggccgatt 9360cattaatgca gctggcacga caggtttccc gactggaaag
cgggcagtga gcgcaacgca 9420attaatgtga gttagctcac tcattaggca
ccccaggctt tacactttat gcttccggct 9480cgtatgttgt gtggaattgt
gagcggataa caatttcaca caggaaacag ctatgaccat 9540gattacgaat
tcgagctcgg taccccacgg aagatccagg tctcgagact aggagacgga
9600tgggaggcgc aacgcgcgat ggggaggggg gcggcgctga cctttctggc
gaggtcgagg 9660tagcgatcga gcagctgcag cgcggacacg atgaggaaga
cgaagatagc cgccatggac 9720atgttcgcca gcggcggcgg agcgaggctg
agccggtctc tccggcctcc ggtcggcgtt 9780aagttgggga tcgtaacgtg
acgtgtctcg tctccacgga tcgacacaac cggcctactc 9840gggtgcacga
cgccgcgata agggcgagat gtccgtgcac gcagcccgtt tggagtcctc
9900gttgcccacg aaccgacccc ttacagaaca aggcctagcc caaaactatt
ctgagttgag 9960cttttgagcc tagcccacct aagccgagcg tcatgaactg
atgaacccac taccactagt 10020caaggcaaac cacaaccaca aatggatcaa
ttgatctaga acaatccgaa ggaggggagg 10080ccacgtcaca ctcacaccaa
ccgaaatatc tgccagaatc agatcaaccg gccaatagga 10140cgccagcgag
cccaacacct ggcgacgccg caaaattcac cgcgaggggc accgggcacg
10200gcaaaaacaa aagcccggcg cggtgagaat atctggcgac tggcggagac
ctggtggcca 10260gcgcgcggcc acatcagcca ccccatccgc ccacctcacc
tccggcgagc caatggcaac 10320tcgtcttaag attccacgag ataaggaccc
gatcgccggc gacgctattt agccaggtgc 10380gccccccacg gtacactcca
ccagcggcat ctatagcaac cggtccagca ctttcacgct 10440cagcttcagc
aagatctacc gtcttcggta cgcgctcact ccgccctctg cctttgttac
10500tgccacgttt ctctgaatgc tctcttgtgt ggtgattgct gagagtggtt
tagctggatc 10560tagaattaca ctctgaaatc gtgttctgcc tgtgctgatt
acttgccgtc ctttgtagca 10620gcaaaatata gggacatggt agtacgaaac
gaagatagaa cctacacagc aatacgagaa 10680atgtgtaatt tggtgcttag
cggtatttat ttaagcacat gttggtgtta tagggcactt 10740ggattcagaa
gtttgctgtt aatttaggca caggcttcat actacatggg tcaatagtat
10800agggattcat attataggcg atactataat aatttgttcg tctgcagagc
ttattatttg 10860ccaaaattag atattcctat tctgtttttg tttgtgtgct
gttaaattgt taacgcctga 10920aggaataaat ataaatgacg aaattttgat
gtttatctct gctcctttat tgtgaccata 10980agtcaagatc agatgcactt
gttttaaata ttgttgtctg aagaaataag tactgacagt 11040attttgatgc
attgatctgc ttgtttgttg taacaaaatt taaaaataaa gagtttcctt
11100tttgttgctc tccttacctc ctgatggtat ctagtatcta ccaactgata
ctatattgct 11160tctctttaca nnnnnntctt gctcgatgcc ttctcctagt
gttgaccagt gttactcaca 11220tagtctttgc tcatttcatt gtaatgcaga
taccaagcgg ttaattaact atgtgcggcg 11280gggccattct cagtgatctc
tactcaccag tgaggcggac ggtcactgcc ggtgacctat 11340ggggagagag
tggcagcagc aagaatgtga agaactggaa aaggagttct tggaagtttg
11400atgaaggcga tgaagacttt gaagctgatt tcaaggattt tgaggattgc
agtagcgagg 11460aggaggtaga ttttggacat gaggaaaaag aattccaatt
gaacagttcg aatttcgtgg 11520aattcaatgg ccatactgcc aaagtcacca
gcaggaagcg aaagatccag taccgaggga 11580tccggcggcg gccttggggc
aaatgggcag cagaaatcag agacccacag aagggcgtcc 11640gagtttggct
tggcacgttc agcactgccg aggaagctgc aagggcatat gacgtggaag
11700ctctacgcat acgtggcaag aaagccaaga tgaatttccc taccaccatc
acagctgctg 11760ggaaacacca ccggcagcgt gtggctcgac cggcaaagaa
gacgtcacaa gagagcctga 11820agtcaagcaa tgcctctggt catgtcatct
cagcaggcag cagtactgat ggcaccgttg 11880tcaagatcga gttgtcacag
tcaccagctt ctccactacc agtgtccagc gcatggcttg 11940atgcttttga
gctgaagcag cttggtggag aaacccctga agctgatggg agagaaaccc
12000ctgaagaaac tgatcatgaa acgggagtga cagcggatat gttttttggc
aatggcgaag 12060tgcggctttc agatgatttt gcgtcttacg agccttaccc
aaattttatg cagttacctt 12120atctagaagg tgactcgtat gaaaacattg
acactctttt caacggtgaa gctgctcagg 12180atggagtgaa catcggaggt
ctttggaatt tcgatgatgt gccaatggac cgtggtgttt 12240actgagcagg
gcgcgccatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc
12300tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta
agcatgtaat 12360aattaacatg taatgcatga cgttatttat gagatgggtt
tttatgatta gagtcccgca 12420attatacatt taatacgcga tagaaaacaa
aatatagcgc gcaaactagg ataaattatc 12480gcgcgcggtg tcatctatgt
tactagatcc gatgataagc tgtcaaacat gaaagcttgg 12540cactggccgt
cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
12600gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
cgcaccgatc 12660gcccttccca acagttgcgc agcctgaatg gcgaatgcta
gagcagcttg agcttggatc 12720agattgtcgt ttcccgcctt cagtttaaac
tatcagtgtt tgacaggata tattggcggg 12780taaacctaag agaaaagagc
gtttattaga ataacggata tttaaaaggg cgtgaaaagg 12840tttatccgtt
cgtccatttg tatgtg 128662813841DNAArtificial SequenceSynthetic
construct pMBXS886modified_base(11171)..(11176)A, T, C or
Gmisc_feature(11171)..(11176)n is a, c, g, or t 28catgccaacc
acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt
cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca
120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt
cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg
gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg
cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg
cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag
gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg
420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac
ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag
cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt
tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac
cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg
cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga
720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg
catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc
caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg
ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga
aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc
gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt
1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc
tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt
ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc
tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat
gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca
tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg
1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg
ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc
gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg
ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt
gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc
tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa
1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga
ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta
tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt
cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc
cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat
gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca
1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg
gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc
ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc
agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc
ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg
tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc
2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga
atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg
ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg
gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg
ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg
tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc
2520gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg
ccgttttccg 2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc
tacgagcttc cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat
ggccagtgtg tgggattacg acctggtact 2700gatggcggtt tcccatctaa
ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc
gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga
2820tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca
ccacgcacgt 2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg
gtgacggtat ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa
gagcgaaacc gggcggccgg agtacatcga 3000gatcgagcta gctgattgga
tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac
cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct
3120ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga
tctacgaacg 3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc
gtgcgcaagc tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga
ggaggcgggg caggctggcc cgatcctagt 3300catgcgctac cgcaacctga
tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg
caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga
3420tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt
acattgggaa 3480cccaaagccg tacattggga accggtcaca catgtaagtg
actgatataa aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa
acttattaaa actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc
agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc
tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc
3720aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg
cgccgtcgcc 3780actcgaccgc cggcgcccac atcaaggcac cctgcctcgc
gcgtttcggt gatgacggtg 3840aaaacctctg acacatgcag ctcccggaga
cggtcacagc ttgtctgtaa gcggatgccg 3900ggagcagaca agcccgtcag
ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960tgacccagtc
acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
4020gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
taaggagaaa 4080ataccgcatc aggcgctctt ccgcttcctc gctcactgac
tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc
tcatagctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg
acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag
agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
4920acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca
gtaaaatata 4980atattttatt ttctcccaat caggcttgat ccccagtaag
tcaaaaaata gctcgacata 5040ctgttcttcc ccgatatcct ccctgatcga
ccggacgcag aaggcaatgt cataccactt 5100gtccgccctg ccgcttctcc
caagatcaat aaagccactt actttgccat ctttcacaaa 5160gatgttgctg
tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt
5220ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt
cccagttttc 5280gcaatccaca tcggccagat cgttattcag taagtaatcc
aattcggcta agcggctgtc 5340taagctattc gtatagggac aatccgatat
gtcgatggag tgaaagagcc tgatgcactc 5400cgcatacagc tcgataatct
tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460gacgccatcg
gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag
5520gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt
cccgttccac 5580atcataggtg gtccctttat accggctgtc cgtcattttt
aaatataggt tttcattttc 5640tcccaccagc ttatatacct tagcaggaga
cattccttcc gtatctttta cgcagcggta 5700tttttcgatc agttttttca
attccggtga tattctcatt ttagccattt attatttcct 5760tcctcttttc
tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc
5820aattcactgt
tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg
5880ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc
gcggtgatca 5940caggcagcaa cgctctgtca tcgttacaat caacatgcta
ccctccgcga gatcatccgt 6000gtttcaaacc cggcagctta gttgccgttc
ttccgaatag catcggtaac atgagcaaag 6060tctgccgcct tacaacggct
ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120cgagtggtga
ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga
6180tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg
cggacgtttt 6240taatgtactg aattaacgcc gaattaattc gggggatctg
gattttagta ctggattttg 6300gttttaggaa ttagaaattt tattgataga
agtattttac aaatacaaat acatactaag 6360ggtttcttat atgctcaaca
catgagcgaa accctatagg aaccctaatt cccttatctg 6420ggaactactc
acacattatt atggagaaac tcgagggatc ccggtcggca tctactctat
6480tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag
tacttctaca 6540cagccatcgg tccagacggc cgcgcttctg cgggcgattt
gtgtacgccc gacagtcccg 6600gctccggatc ggacgattgc gtcgcatcga
ccctgcgccc aagctgcatc atcgaaattg 6660ccgtcaacca agctctgata
gagttggtca agaccaatgc ggagcatata cgcccggagc 6720cgcggcgatc
ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
6780caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga
atccccgaac 6840atcgcctcgc tccagtcaat gaccgctgtt atgcggccat
tgtccgtcag gacattgttg 6900gagccgaaat ccgcgtgcac gaggtgccgg
acttcggggc agtcctcggc ccaaagcatc 6960agctcatcga gagcctgcgc
gacggacgca ctgacggtgt cgtccatcac agtttgccag 7020tgatacacat
ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
7080attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc
cgcagcgatc 7140gcatccatgg cctccgcgac cggctgcagt tatcatcatc
atcatagaca cacgaaataa 7200agtaatcaga ttatcagtta aagctatgta
atatttacac cataaccaat caattaaaaa 7260atagatcagt ttaaagaaag
atcaaagctc aaaaaaataa aaagagaaaa gggtcctaac 7320caagaaaatg
aaggagaaaa actagaaatt tacctgcaga acagcgggca gttcggtttc
7380aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg
tcaggctctc 7440gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
gcggccgatg caaagtgccg 7500ataaacataa cgatctttgt agaaaccatc
ggcgcagcta tttacccgca ggacatatcc 7560acgccctcct acatcgaagc
tgaaagcacg agattcttcg ccctccgaga gctgcatcag 7620gtcggagacg
ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc
7680aggctttttc atggtagagg agctcgccgc ttggtatctg cattacaatg
aaatgagcaa 7740agactatgtg agtaacactg gtcaacacta gggagaaggc
atcgagcaag atacgtatgt 7800aaagagaagc aatatagtgt cagttggtag
atactagata ccatcaggag gtaaggagag 7860caacaaaaag gaaactcttt
atttttaaat tttgttacaa caaacaagca gatcaatgca 7920tcaaaatact
gtcagtactt atttcttcag acaacaatat ttaaaacaag tgcatctgat
7980cttgacttat ggtcacaata aaggagcaga gataaacatc aaaatttcgt
catttatatt 8040tattccttca ggcgttaaca atttaacagc acacaaacaa
aaacagaata ggaatatcta 8100attttggcaa ataataagct ctgcagacga
acaaattatt atagtatcgc ctataatatg 8160aatccctata ctattgaccc
atgtagtatg aagcctgtgc ctaaattaac agcaaacttc 8220tgaatccaag
tgccctataa caccaacatg tgcttaaata aataccgcta agcaccaaat
8280tacacatttc tcgtattgct gtgtaggttc tatcttcgtt tcgtactacc
atgtccctat 8340attttgctgc tacaaaggac ggcaagtaat cagcacaggc
agaacacgat ttcagagtgt 8400aattctagat ccagctaaac cactctcagc
aatcaccaca caagagagca ttcagagaaa 8460cgtggcagta acaaaggcag
agggcggagt gagcgcgtac cgaagacggt agatctctcg 8520agagagatag
atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg
8580aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt
catcccttac 8640gtcagtggag atatcacatc aatccacttg ctttgaagac
gtggttggaa cgtcttcttt 8700ttccacgatg ctcctcgtgg gtgggggtcc
atctttggga ccactgtcgg cagaggcatc 8760ttgaacgata gcctttcctt
tatcgcaatg atggcatttg taggtgccac cttccttttc 8820tactgtcctt
ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga
8880tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg
tatctttgat 8940attcttggag tagacgagag tgtcgtgctc caccatgtta
tcacatcaat ccacttgctt 9000tgaagacgtg gttggaacgt cttctttttc
cacgatgctc ctcgtgggtg ggggtccatc 9060tttgggacca ctgtcggcag
aggcatcttg aacgatagcc tttcctttat cgcaatgatg 9120gcatttgtag
gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg
9180gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct
caatagccct 9240ttggtcttct gagactgtat ctttgatatt cttggagtag
acgagagtgt cgtgctccac 9300catgttggca agctgctcta gccaatacgc
aaaccgcctc tccccgcgcg ttggccgatt 9360cattaatgca gctggcacga
caggtttccc gactggaaag cgggcagtga gcgcaacgca 9420attaatgtga
gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
9480cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
ctatgaccat 9540gattacgaat tcgagctcgg taccccacgg aagatccagg
tctcgagact aggagacgga 9600tgggaggcgc aacgcgcgat ggggaggggg
gcggcgctga cctttctggc gaggtcgagg 9660tagcgatcga gcagctgcag
cgcggacacg atgaggaaga cgaagatagc cgccatggac 9720atgttcgcca
gcggcggcgg agcgaggctg agccggtctc tccggcctcc ggtcggcgtt
9780aagttgggga tcgtaacgtg acgtgtctcg tctccacgga tcgacacaac
cggcctactc 9840gggtgcacga cgccgcgata agggcgagat gtccgtgcac
gcagcccgtt tggagtcctc 9900gttgcccacg aaccgacccc ttacagaaca
aggcctagcc caaaactatt ctgagttgag 9960cttttgagcc tagcccacct
aagccgagcg tcatgaactg atgaacccac taccactagt 10020caaggcaaac
cacaaccaca aatggatcaa ttgatctaga acaatccgaa ggaggggagg
10080ccacgtcaca ctcacaccaa ccgaaatatc tgccagaatc agatcaaccg
gccaatagga 10140cgccagcgag cccaacacct ggcgacgccg caaaattcac
cgcgaggggc accgggcacg 10200gcaaaaacaa aagcccggcg cggtgagaat
atctggcgac tggcggagac ctggtggcca 10260gcgcgcggcc acatcagcca
ccccatccgc ccacctcacc tccggcgagc caatggcaac 10320tcgtcttaag
attccacgag ataaggaccc gatcgccggc gacgctattt agccaggtgc
10380gccccccacg gtacactcca ccagcggcat ctatagcaac cggtccagca
ctttcacgct 10440cagcttcagc aagatctacc gtcttcggta cgcgctcact
ccgccctctg cctttgttac 10500tgccacgttt ctctgaatgc tctcttgtgt
ggtgattgct gagagtggtt tagctggatc 10560tagaattaca ctctgaaatc
gtgttctgcc tgtgctgatt acttgccgtc ctttgtagca 10620gcaaaatata
gggacatggt agtacgaaac gaagatagaa cctacacagc aatacgagaa
10680atgtgtaatt tggtgcttag cggtatttat ttaagcacat gttggtgtta
tagggcactt 10740ggattcagaa gtttgctgtt aatttaggca caggcttcat
actacatggg tcaatagtat 10800agggattcat attataggcg atactataat
aatttgttcg tctgcagagc ttattatttg 10860ccaaaattag atattcctat
tctgtttttg tttgtgtgct gttaaattgt taacgcctga 10920aggaataaat
ataaatgacg aaattttgat gtttatctct gctcctttat tgtgaccata
10980agtcaagatc agatgcactt gttttaaata ttgttgtctg aagaaataag
tactgacagt 11040attttgatgc attgatctgc ttgtttgttg taacaaaatt
taaaaataaa gagtttcctt 11100tttgttgctc tccttacctc ctgatggtat
ctagtatcta ccaactgata ctatattgct 11160tctctttaca nnnnnntctt
gctcgatgcc ttctcctagt gttgaccagt gttactcaca 11220tagtctttgc
tcatttcatt gtaatgcaga taccaagcgg ttaattaact atgtgcggcg
11280gggccattct cagtgatctc tactcaccag tgaggcggac ggtcactgcc
ggtgacctat 11340ggggagagag tggcagcagc aagaatgtga agaactggaa
aaggagttct tggaagtttg 11400atgaaggcga tgaagacttt gaagctgatt
tcaaggattt tgaggattgc agtagcgagg 11460aggaggtaga ttttggacat
gaggaaaaag aattccaatt gaacagttcg aatttcgtgg 11520aattcaatgg
ccatactgcc aaagtcacca gcaggaagcg aaagatccag taccgaggga
11580tccggcggcg gccttggggc aaatgggcag cagaaatcag agacccacag
aagggcgtcc 11640gagtttggct tggcacgttc agcactgccg aggaagctgc
aagggcatat gacgtggaag 11700ctctacgcat acgtggcaag aaagccaaga
tgaatttccc taccaccatc acagctgctg 11760ggaaacacca ccggcagcgt
gtggctcgac cggcaaagaa gacgtcacaa gagagcctga 11820agtcaagcaa
tgcctctggt catgtcatct cagcaggcag cagtactgat ggcaccgttg
11880tcaagatcga gttgtcacag tcaccagctt ctccactacc agtgtccagc
gcatggcttg 11940atgcttttga gctgaagcag cttggtggag aaacccctga
agctgatggg agagaaaccc 12000ctgaagaaac tgatcatgaa acgggagtga
cagcggatat gttttttggc aatggcgaag 12060tgcggctttc agatgatttt
gcgtcttacg agccttaccc aaattttatg cagttacctt 12120atctagaagg
tgactcgtat gaaaacattg acactctttt caacggtgaa gctgctcagg
12180atggagtgaa catcggaggt ctttggaatt tcgatgatgt gccaatggac
cgtggtgttt 12240actgaatgtg cggcggggcc attctcagtg atctctactc
accagtgagg cggacggtca 12300ctgccggtga cctatgggga gagagtggca
gcagcaagaa tgtgaagaac tggaaaagga 12360gttcttggaa gtttgatgaa
ggcgatgaag actttgaagc tgatttcaag gattttgagg 12420attgcagtag
cgaggaggag gtagattttg gacatgagga aaaagaattc caattgaaca
12480gttcgaattt cgtggaattc aatggccata ctgccaaagt caccagcagg
aagcgaaaga 12540tccagtaccg agggatccgg cggcggcctt ggggcaaatg
ggcagcagaa atcagagacc 12600cacagaaggg cgtccgagtt tggcttggca
cgttcagcac tgccgaggaa gctgcaaggg 12660catatgacgt ggaagctcta
cgcatacgtg gcaagaaagc caagatgaat ttccctacca 12720ccatcacagc
tgctgggaaa caccaccggc agcgtgtggc tcgaccggca aagaagacgt
12780cacaagagag cctgaagtca agcaatgcct ctggtcatgt catctcagca
ggcagcagta 12840ctgatggcac cgttgtcaag atcgagttgt cacagtcacc
agcttctcca ctaccagtgt 12900ccagcgcatg gcttgatgct tttgagctga
agcagcttgg tggagaaacc cctgaagctg 12960atgggagaga aacccctgaa
gaaactgatc atgaaacggg agtgacagcg gatatgtttt 13020ttggcaatgg
cgaagtgcgg ctttcagatg attttgcgtc ttacgagcct tacccaaatt
13080ttatgcagtt accttatcta gaaggtgact cgtatgaaaa cattgacact
cttttcaacg 13140gtgaagctgc tcaggatgga gtgaacatcg gaggtctttg
gaatttcgat gatgtgccaa 13200tggaccgtgg tgtttactga gcagggcgcg
ccatcgttca aacatttggc aataaagttt 13260cttaagattg aatcctgttg
ccggtcttgc gatgattatc atataatttc tgttgaatta 13320cgttaagcat
gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat
13380gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
agcgcgcaaa 13440ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta
gatccgatga taagctgtca 13500aacatgaaag cttggcactg gccgtcgttt
tacaacgtcg tgactgggaa aaccctggcg 13560ttacccaact taatcgcctt
gcagcacatc cccctttcgc cagctggcgt aatagcgaag 13620aggcccgcac
cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgctagagca
13680gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca
gtgtttgaca 13740ggatatattg gcgggtaaac ctaagagaaa agagcgttta
ttagaataac ggatatttaa 13800aagggcgtga aaaggtttat ccgttcgtcc
atttgtatgt g 138412987PRTPanicum virgatum 29Val Thr Ser Arg Lys Arg
Lys Ile Gln Tyr Arg Gly Ile Arg Arg Arg1 5 10 15Pro Trp Gly Lys Trp
Ala Ala Glu Ile Arg Asp Pro Gln Lys Gly Val 20 25 30Arg Val Trp Leu
Gly Thr Phe Ser Thr Ala Glu Glu Ala Ala Arg Ala 35 40 45Tyr Asp Val
Glu Ala Leu Arg Ile Arg Gly Lys Lys Ala Lys Met Asn 50 55 60Phe Pro
Thr Thr Ile Thr Ala Ala Gly Lys His His Arg Gln Arg Val65 70 75
80Ala Arg Pro Ala Lys Lys Thr 853087PRTSetaria italica 30Val Ala
Arg Arg Lys Arg Lys Thr Gln Tyr Arg Gly Ile Arg Arg Arg1 5 10 15Pro
Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Cys Lys Gly Val 20 25
30Arg Val Trp Leu Gly Thr Tyr Asn Thr Ala Glu Glu Ala Ala Arg Ala
35 40 45Tyr Asp Val Ala Ala Arg Arg Ile Arg Gly Lys Lys Ala Lys Val
Asn 50 55 60Phe Pro Asp Thr Ile Thr Ala Ser Ala Lys Arg Leu Pro Gly
Arg Val65 70 75 80Pro Arg Pro Ala Lys Lys Val 853187PRTSorghum
bicolor 31Val Ala Ser Arg Lys Arg Arg Thr Gln Tyr Arg Gly Ile Arg
Arg Arg1 5 10 15Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Arg
Lys Gly Val 20 25 30Arg Val Trp Leu Gly Thr Tyr Ser Thr Ala Glu Glu
Ala Ala Arg Ala 35 40 45Tyr Asp Thr Ala Ala Trp Arg Ile Arg Gly Lys
Lys Ala Lys Val Asn 50 55 60Phe Pro Ser Ala Ile Thr Asn Pro Glu Lys
Arg Arg Arg Gly Arg Val65 70 75 80Ala Arg Pro Arg Lys Lys Ile
853294PRTOryza sativa 32Gly Gly Ser Arg Lys Arg Lys Thr Arg Tyr Arg
Gly Ile Arg Gln Arg1 5 10 15Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg
Asp Pro Arg Lys Gly Val 20 25 30Arg Val Trp Leu Gly Thr Phe Gly Thr
Ala Glu Glu Ala Ala Met Ala 35 40 45Tyr Asp Val Glu Ala Arg Arg Ile
Arg Gly Lys Lys Ala Lys Val Asn 50 55 60Phe Pro Asp Ala Ala Ala Ala
Ala Pro Lys Arg Pro Arg Arg Ser Ser65 70 75 80Ala Lys His Ser Pro
Gln Gln Gln Lys Ala Arg Ser Ser Ser 85 9033110PRTJatropha curcas
33Phe Asn Gly Gln Ala Glu Lys Ser Ala Lys Arg Lys Arg Lys Asn Gln1
5 10 15Tyr Arg Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu
Ile 20 25 30Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Phe
Asn Thr 35 40 45Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg
Arg Ile Arg 50 55 60Gly Lys Lys Ala Lys Val Asn Phe Pro Glu Glu Ala
Pro His Ala Ser65 70 75 80Pro Lys Arg Pro Ser Lys Ala Asn Ser Gln
Lys Ser Leu Gly Lys Thr 85 90 95Asn Leu Ala Glu Asn Leu Asn Tyr Leu
Asp Asn Pro Glu Gln 100 105 11034110PRTPopulus trichocarpa 34Phe
Ser Gly Pro Ala Glu Lys Ser Ala Lys Arg Lys Arg Lys Asn Gln1 5 10
15Phe Arg Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile
20 25 30Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn
Thr 35 40 45Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ser Glu Ala Arg Arg
Ile Arg 50 55 60Gly Lys Lys Ala Lys Val Asn Phe Pro Asp Glu Ala Pro
Cys Ala Ser65 70 75 80Ala Arg His Pro Ile Lys Glu Asn Ser Gln Lys
Arg Leu Thr Lys Ala 85 90 95Asn Leu Ser Gln Asp Phe Ser Tyr Leu Ser
Asn Pro Glu Thr 100 105 11035117PRTGossypium hirsutum 35Phe Asn Gly
Gln Ala Glu Lys Cys Ala Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg
Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg
Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn Thr 35 40
45Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg
50 55 60Gly Lys Lys Ala Lys Val Asn Phe Pro Asn Glu Thr Pro Arg Thr
Ser65 70 75 80Pro Lys His Ala Val Lys Thr Asn Ser Gln Lys Pro Leu
Ser Lys Ser 85 90 95Asn Ser Ser Pro Val Gln Pro Asn Leu Asn Gln Asn
Tyr Asn Tyr Leu 100 105 110Asn Gln Pro Glu Gln 11536117PRTTheobroma
cacao 36Phe Asn Gly Gln Ala Glu Lys Ser Ala Lys Arg Lys Arg Lys Asn
Gln1 5 10 15Tyr Arg Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala
Glu Ile 20 25 30Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr
Phe Asn Thr 35 40 45Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala
Arg Arg Ile Arg 50 55 60Gly Lys Lys Ala Lys Val Asn Phe Pro Asp Glu
Ala Pro Arg Thr Ser65 70 75 80Pro Lys Arg Ala Val Lys Ala Asn Ser
Gln Lys Ser Leu Ser Arg Ser 85 90 95Asn Leu Ser Pro Val Gln Pro Asn
Leu Asp Gln Asn Phe Asn Tyr Leu 100 105 110Ser Lys Pro Glu Gln
11537117PRTMalus domestica 37Phe Asp Gly Gln Ala Glu Lys Ser Ala
Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln Arg Pro
Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg Lys Gly Val
Arg Val Trp Leu Gly Thr Phe Asn Thr 35 40 45Ala Glu Glu Ala Ala Arg
Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys Ala Lys
Val Asn Phe Pro Glu Glu Thr Pro Cys Ala Ser65 70 75 80Ala Lys Arg
Ser Ile Lys Glu Asn Pro Gln Lys Leu Ile Ala Lys Thr 85 90 95Asn Leu
Asn Gly Thr Gln Ser Asn Pro Asn Gln Asn Phe Asn Phe Val 100 105
110Asn Asp Ser Ser Glu 11538118PRTMorus alba 38Ser Asp Gly Gln Ala
Glu Lys Ser Ala Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile
Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro
Arg Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn Thr 35 40 45Ala Glu
Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly
Lys Lys Ala Lys Val Asn Phe Pro Asp Glu Thr Pro Arg Ala Leu65 70 75
80Pro Lys His Pro Val Lys Glu Ser Pro Lys Arg Ser Leu Pro Lys Glu
85 90 95Asn Ser Asn Ser Thr Glu Ser Asn Leu Asn Asn Gln Ser Phe Asn
Ser 100 105 110Val Asn Asn Ser Asp Leu 11539105PRTCucumis sativus
39Phe Asn Glu Gln Ala Glu Lys Ser Ala Asn Thr Lys Arg Lys Asn Gln1
5 10 15Tyr Arg Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu
Ile 20 25 30Arg Asp Pro Arg Lys Gly Ala Arg Val Trp Leu Gly Thr Phe
Asn Thr 35 40 45Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg
Arg Ile Arg 50 55 60Gly Asn Lys Ala Arg Val Asn Phe Pro Asp Glu Pro
Leu Pro Asn Thr65 70 75 80Gln Lys Arg Lys Asn Ser Gln Lys Ser
Lys
Gln His Ile Lys Glu Asn 85 90 95Val Lys Ala Asn Gln His Pro Asn Gln
100 10540113PRTSolanum lycopersicum 40Ser Asn Cys Glu Ala Asp Arg
Ser Ser Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg Lys
Gly Ile Arg Val Trp Leu Gly Thr Phe Asn Ser 35 40 45Ala Glu Glu Ala
Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys
Ala Lys Val Asn Phe Pro Asp Glu Ala Pro Val Ser Val65 70 75 80Ser
Arg Arg Ala Ile Lys Gln Asn Pro Gln Lys Ala Leu Arg Glu Glu 85 90
95Thr Leu Asn Thr Val Gln Pro Asn Met Thr Tyr Ile Ser Asn Leu Asp
100 105 110Gly41113PRTCapsicum annuum 41Ser Ser Cys Asp Thr Glu Lys
Ser Ser Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg Lys
Gly Ile Arg Val Trp Leu Gly Thr Phe Asn Ser 35 40 45Ala Glu Glu Ala
Ala Arg Ala Tyr Asp Val Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys
Ala Lys Val Asn Phe Pro Asp Gly Ser Pro Ala Ser Ala65 70 75 80Ser
Arg Arg Ala Val Lys Pro Asn Pro Gln Glu Ala Leu Arg Glu Glu 85 90
95Ile Leu Asn Thr Val Gln Pro Asn Thr Thr Tyr Ile Asn Asn Leu Asp
100 105 110Gly42113PRTNicotiana tabacum 42Ser Asp Lys Asp Ala Asp
Arg Ser Ser Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg
Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg
Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn Thr 35 40 45Ala Glu Glu
Ala Ala Arg Ala Tyr Asp Val Glu Ala Arg Arg Ile Arg 50 55 60Gly Asn
Lys Ala Lys Val Asn Phe Pro Asp Glu Ala Pro Val Pro Ala65 70 75
80Ser Arg Arg Thr Val Lys Val Asn Pro Gln Lys Val Leu Pro Lys Glu
85 90 95Ile Leu Asp Ser Val Gln Pro Asp Ser Thr Ile Ile Asn Asn Met
Glu 100 105 110Asp43108PRTGlycine max 43Phe Gln Gly Arg Ala Glu Ile
Ser Ala Asn Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg Lys
Gly Val Arg Val Trp Leu Gly Thr Phe Asn Thr 35 40 45Ala Glu Glu Ala
Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys
Ala Lys Val Asn Phe Pro Glu Ala Pro Gly Thr Ser Ser65 70 75 80Val
Lys Arg Ser Lys Val Asn Pro Gln Glu Asn Leu Lys Thr Val Gln 85 90
95Pro Asn Leu Gly His Lys Phe Ser Ala Gly Asn Asn 100
10544105PRTArachis hypogaea 44Val Lys Ala Gln Ser Glu Lys Ser Ala
Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln Arg Pro
Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Arg Lys Gly Val
Arg Val Trp Leu Gly Thr Phe Ser Thr 35 40 45Ala Glu Glu Ala Ala Arg
Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys Ala Lys
Val Asn Phe Pro Glu Glu Ala Pro Arg Thr Pro65 70 75 80Pro Lys Arg
Ala Arg Pro Asn Leu Asn Ala Val Gln Pro Asn Leu Ser 85 90 95His Lys
Phe Ser Val Gly Asn Asn Met 100 10545108PRTMedicago truncatula
45Ser Lys Ser Asn Glu Gln Gly Glu Lys Glu Leu Lys Arg Lys Arg Lys1
5 10 15Asn Gln Tyr Arg Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala
Ala 20 25 30Glu Ile Arg Asp Pro Arg Lys Gly Val Arg Val Trp Leu Gly
Thr Phe 35 40 45Asn Thr Ala Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu
Ala Arg Arg 50 55 60Ile Arg Gly Lys Lys Ala Lys Val Asn Phe Pro Glu
Glu Ala Pro Asn65 70 75 80Ala Ser Ser Lys Arg Leu Lys Thr Asn Ser
Glu Thr Gln Leu Leu Asp 85 90 95Lys Asn Leu Asn Ser Phe Lys Cys Glu
Asn Ile Glu 100 10546114PRTZea mays 46Tyr Asp Ala Pro Ala Ala Arg
Leu Ala Lys Arg Lys Arg Lys Asn Gln1 5 10 15Tyr Arg Gly Ile Arg Gln
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp Pro Gln Lys
Gly Val Arg Val Trp Leu Gly Thr Phe Asn Ser 35 40 45Pro Glu Glu Ala
Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55 60Gly Lys Lys
Ala Lys Val Asn Phe Pro Asp Ala Pro Ala Val Gly Gln65 70 75 80Lys
Cys Arg Ser Ser Ser Ala Ser Ala Lys Ala Leu Lys Ser Cys Val 85 90
95Glu Gln Lys Pro Ile Val Lys Thr Asp Met Asn Ile Leu Ala Asn Thr
100 105 110Asn Ala47114PRTBrachypodium distachyon 47Phe Asp Gly Pro
Ala Glu Arg Ser Ala Lys Arg Lys Arg Lys Asn Gln1 5 10 15Phe Arg Gly
Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile 20 25 30Arg Asp
Pro Asn Lys Gly Val Arg Val Trp Leu Gly Thr Phe Asn Ser 35 40 45Ala
Glu Glu Ala Ala Arg Ala Tyr Asp Ala Glu Ala Arg Arg Ile Arg 50 55
60Gly Asn Lys Ala Lys Val Asn Phe Pro Glu Glu Pro Arg Ala Ala Gln65
70 75 80Lys Arg Arg Ala Gly Pro Ala Ala Ala Lys Val Pro Lys Ser Arg
Val 85 90 95Glu Gln Lys Pro Asn Val Lys Pro Ala Val Asn Asn Leu Ala
Asn Thr 100 105 110Asn Ala48108PRTTriticum aestivum 48Asp Asp Asp
Cys Ala Ser Gly Ser Ala Arg Lys Arg Lys Asn Gln Phe1 5 10 15Arg Gly
Ile Arg Arg Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg 20 25 30Asp
Pro Arg Lys Gly Val Arg Val Trp Leu Gly Thr Tyr Asn Ser Ala 35 40
45Glu Glu Ala Ala Arg Ala Tyr Asp Val Glu Ala Arg Arg Ile Arg Gly
50 55 60Lys Lys Ala Glu Val Asn Phe Pro Glu Glu Ala Pro Met Ala Pro
Gln65 70 75 80Gln Arg Cys Ala Thr Ala Val Lys Val Pro Glu Phe Asn
Thr Glu Gln 85 90 95Lys Pro Val Leu Asn Thr Met Gly Asn Ala Asp Val
100 10549102PRTHordeum vulgare 49Tyr Asp Gly Gly Arg Ala Ala His
Ala Ala Ser Arg Lys Lys Arg Thr1 5 10 15Gly His Leu His Gly Ile Arg
Gln Arg Pro Trp Gly Lys Trp Ala Ala 20 25 30Glu Ile Arg Asp Pro His
Lys Gly Thr Arg Val Trp Leu Gly Thr Phe 35 40 45Asp Thr Ala Asp Asp
Ala Ala Arg Ala Tyr Asp Val Ala Ala Arg Arg 50 55 60Leu Arg Gly Ser
Lys Ala Lys Val Asn Phe Pro Asp Ala Ala Arg Thr65 70 75 80Gly Ala
Arg Pro Arg Arg Ala Ser Arg Arg Thr Ala Gln Lys Pro Gln 85 90 95Cys
Pro Pro Ala Arg Thr 1005092PRTZea mays 50Thr Leu Thr Thr Thr Met
Arg His Tyr Arg Gly Val Arg Arg Arg Pro1 5 10 15Trp Gly Lys Trp Ala
Ala Glu Ile Arg Asp Pro Ala Lys Ala Ala Arg 20 25 30Val Trp Leu Gly
Thr Phe Asp Thr Ala Glu Ala Ala Ala Ala Ala Tyr 35 40 45Asp Arg Ala
Ala Leu Gln Phe Lys Gly Ala Lys Ala Lys Leu Asn Phe 50 55 60Pro Glu
Arg Val Arg Gly Arg Thr Gly Gln Gly Ala Phe Leu Val Ser65 70 75
80Pro Gly Ile Pro Gln Pro Pro Pro Val Ser Ala Pro 85
905196PRTSorghum bicolor 51Thr Ser Thr Thr Thr Met Arg His Tyr Arg
Gly Val Arg Arg Arg Pro1 5 10 15Trp Gly Lys Trp Ala Ala Glu Ile Arg
Asp Pro Ala Lys Ala Ala Arg 20 25 30Val Trp Leu Gly Thr Phe Asp Thr
Ala Glu Ala Ala Ala Ala Ala Tyr 35 40 45Asp Asp Ala Ala Leu Arg Phe
Lys Gly Ala Lys Ala Lys Leu Asn Phe 50 55 60Pro Glu Arg Val Arg Gly
Arg Thr Gly Gln Gly Ala Phe Leu Val Ser65 70 75 80Pro Gly Ile Pro
Gln Pro Pro Pro Pro Pro Val Ser Ala Pro Pro Leu 85 90
955291PRTPanicum virgatum 52Tyr Gly Thr Arg Met His Tyr Arg Gly Val
Arg Arg Arg Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile Arg Asp Pro
Ala Lys Ala Ala Arg Val Trp 20 25 30Leu Gly Thr Phe Asp Thr Ala Glu
Ala Ala Ala Ala Ala Tyr Asp Asp 35 40 45Ala Ala Leu Arg Phe Lys Gly
Ala Lys Ala Lys Leu Asn Phe Pro Glu 50 55 60Arg Val Arg Gly Arg Thr
Gly Gln Gly Ala Phe Leu Val Ser Pro Gly65 70 75 80Val Pro Gln Gln
Pro Pro Pro Ser Ser Leu Pro 85 9053100PRTHordeum vulgare 53Gly Arg
Lys Arg His Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys1 5 10 15Trp
Ala Ala Glu Ile Arg Asp Pro Lys Lys Ala Ala Arg Val Trp Leu 20 25
30Gly Thr Phe Asp Thr Ala Glu Asp Ala Ala Ile Ala Tyr Asp Glu Ala
35 40 45Ala Leu Arg Phe Lys Gly Thr Lys Ala Lys Leu Asn Phe Pro Glu
Arg 50 55 60Val Gln Gly Arg Thr Asp Leu Gly Phe Val Val Thr Arg Gly
Ile Pro65 70 75 80Asp Arg Leu Gln Gln Gln Gln His Tyr Pro Ala Ala
Val Gly Ala Pro 85 90 95Ala Met Arg Pro 1005499PRTBrachypodium
distachyon 54Gly Arg Lys Arg His Tyr Arg Gly Val Arg Gln Arg Pro
Trp Gly Lys1 5 10 15Trp Ala Ala Glu Ile Arg Asp Pro Lys Lys Ala Ala
Arg Val Trp Leu 20 25 30Gly Thr Phe Asp Thr Ala Glu Asp Ala Ala Ile
Ala Tyr Asp Glu Ala 35 40 45Ala Leu Arg Phe Lys Gly Thr Lys Ala Lys
Leu Asn Phe Pro Glu Arg 50 55 60Val Gln Gly Arg Thr Asp Leu Gly Phe
Val Val Thr Arg Gly Ile Pro65 70 75 80Asp Arg Ser Ser Leu His His
Gln Gln His Tyr Pro Gly Ser Thr Ala 85 90 95Met Arg
Pro55101PRTOryza sativa 55Gly Arg Arg Arg His Tyr Arg Gly Val Arg
Gln Arg Pro Trp Gly Lys1 5 10 15Trp Ala Ala Glu Ile Arg Asp Pro Lys
Lys Ala Ala Arg Val Trp Leu 20 25 30Gly Thr Phe Asp Thr Ala Glu Asp
Ala Ala Ile Ala Tyr Asp Glu Ala 35 40 45Ala Leu Arg Phe Lys Gly Thr
Lys Ala Lys Leu Asn Phe Pro Glu Arg 50 55 60Val Gln Gly Arg Thr Asp
Leu Gly Phe Leu Val Thr Arg Gly Ile Pro65 70 75 80Pro Ala Ala Thr
His Gly Gly Gly Tyr Tyr Pro Ser Ser Ser Pro Ala 85 90 95Ala Gly Ala
Cys Pro 10056105PRTJatropha curcas 56Asn Thr Arg Arg Arg His Tyr
Arg Gly Val Arg Gln Arg Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile
Arg Asp Pro Lys Lys Ala Ala Arg Val Trp 20 25 30Leu Gly Thr Phe Asp
Thr Ala Glu Asp Ala Ala Leu Ala Tyr Asp Lys 35 40 45Ala Ala Leu Lys
Phe Lys Gly Thr Lys Ala Lys Leu Asn Phe Pro Glu 50 55 60Arg Val Gln
Gly Lys Pro Glu Phe Ser Tyr Phe Met Thr Ser Ser Gly65 70 75 80Asp
Ser Ser Ser Ala Leu Ala Pro Glu Gln Asn Pro Met Ala Ala Ala 85 90
95Ala Ser Ala Pro Ser Arg His Tyr Leu 100 10557101PRTPopulus
trichocarpa 57Asn Thr Arg Arg Arg His Tyr Arg Gly Val Arg Gln Arg
Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile Arg Asp Pro Lys Lys Ala
Ala Arg Val Trp 20 25 30Leu Gly Thr Phe Asp Thr Ala Glu Asp Ala Ala
Val Ala Tyr Asp Lys 35 40 45Ala Ala Leu Lys Phe Lys Gly Thr Lys Ala
Lys Leu Asn Phe Pro Glu 50 55 60Arg Val Gln Gly Arg Thr Glu Phe Gly
Tyr Tyr Met Gly Ser Gly Thr65 70 75 80Ser Thr Asn Val Leu Thr Glu
Gln Ser Pro Arg Pro Val Ala Pro Pro 85 90 95Pro Pro Pro Pro Pro
1005897PRTTheobroma cacao 58Glu Glu Asn Thr Arg Arg Arg His Tyr Arg
Gly Val Arg Gln Arg Pro1 5 10 15Trp Gly Lys Trp Ala Ala Glu Ile Arg
Asp Pro Lys Lys Ala Ala Arg 20 25 30Val Trp Leu Gly Thr Phe Asp Thr
Ala Glu Asp Ala Ala Leu Ala Tyr 35 40 45Asp Arg Ala Ala Leu Lys Phe
Lys Gly Thr Lys Ala Lys Leu Asn Phe 50 55 60Pro Glu Arg Val Gln Gly
Asn Thr Glu Val Ser Tyr Phe Thr Gly His65 70 75 80Gly Asp Ser Ser
Thr Val Arg Pro Asp Gln Asn Pro Thr Pro Ala Ala 85 90
95Thr5987PRTMedicago truncatula 59Thr Lys Lys Lys Pro His Tyr Arg
Gly Val Arg Gln Arg Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile Arg
Asp Pro Lys Lys Ala Ala Arg Val Trp 20 25 30Leu Gly Thr Phe Asp Thr
Ala Glu Asp Ala Ala Leu Ala Tyr Asp Lys 35 40 45Ala Ala Leu Lys Phe
Lys Gly Thr Lys Ala Lys Leu Asn Phe Pro Glu 50 55 60Arg Val Val Gln
Cys Asn Ser Tyr Ser Ser Thr Ala Asn Asn Ala Ile65 70 75 80Gln Gln
Ser Asp Tyr Val Ser 8560110PRTGlycine max 60Val Thr Lys Lys Pro His
Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu
Ile Arg Asp Pro Lys Lys Ala Ala Arg Val Trp 20 25 30Leu Gly Thr Phe
Glu Thr Ala Glu Asp Ala Ala Leu Ala Tyr Asp Lys 35 40 45Ala Ala Leu
Lys Phe Lys Gly Thr Lys Ala Lys Leu Asn Phe Pro Glu 50 55 60Arg Leu
His Gln Asn Val Pro Tyr Met Gln Gln His Gln Gln Gly Ser65 70 75
80Ser Asn Arg Asn Val Phe Pro Phe His Ala Thr Ser Ser Thr Ser Ser
85 90 95Ser Ala Thr Gly Ser Val Ser Ser Leu Asp Ala Val Ala Pro 100
105 11061108PRTMalus domestica 61Thr Val Arg Arg Arg His Tyr Arg
Gly Val Arg Gln Arg Pro Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile Arg
Asp Pro Lys Lys Ala Ala Arg Val Trp 20 25 30Leu Gly Thr Phe Glu Thr
Ala Glu Asp Ala Ala Ile Ala Tyr Asp Asn 35 40 45Ala Ala Leu Arg Phe
Lys Gly Thr Lys Ala Lys Leu Asn Phe Pro Glu 50 55 60Arg Val Gln Gly
Lys Thr Asp Phe Gly Ile Leu Met Gly Ser Ser Gly65 70 75 80Thr Thr
Thr Asn Ser Ser Ser Gly Ala Ala Ser Thr Gln Arg Thr Gln 85 90 95Asn
Leu Met Arg Pro Ala Gly Gln Thr Ala Pro Ala 100 1056292PRTCapsicum
annuum 62Gly Ser Gly Arg Arg His Tyr Arg Gly Val Arg Gln Arg Pro
Trp Gly1 5 10 15Lys Trp Ala Ala Glu Ile Arg Asn Pro Lys Lys Ala Ala
Arg Val Trp 20 25 30Leu Gly Thr Phe Asp Arg Ala Glu Asp Ala Ala Leu
Ala Tyr Asp Glu 35 40 45Ala Ala Val Arg Phe Lys Gly Ser Lys Ala Lys
Leu Asn Phe Pro Glu 50 55 60Arg Leu Val Gln Gly Gln Pro Gln Leu Leu
Ser Gln Asp Thr Ser Pro65 70 75 80Gln His Asn Ser His His Phe Glu
Glu Phe Asn Thr 85 9063107PRTBrassica juncea 63Ser Gly Asp Gly Pro
Gln Arg Arg Tyr Arg Gly Val Arg Gln Arg Pro1 5 10
15Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Phe Lys Ala Ala Arg
20 25 30Val Trp Leu Gly Thr Phe Asp Asn Ala Glu Ser Ala Ala Arg Ala
Tyr 35 40 45Asp Glu Ala Ala Leu Arg Phe Arg Gly Asn Lys Ala Lys Leu
Asn Phe 50 55 60Pro Glu Asn Val Lys Leu Val Arg Pro Ala Ser Thr Thr
Pro Thr Leu65 70 75 80Ser Val Pro Gln Thr Ala Val Gln Arg Pro Thr
Gln Leu Arg Asn Ser 85 90 95Gly Ser Thr Ser Thr Ile Leu Pro Val Arg
His 100 10564101PRTSolanum lycopersicum 64Asn Asn Glu Lys Arg Arg
Arg Gln Tyr Arg Gly Val Arg Gln Arg Pro1 5 10 15Trp Gly Lys Trp Ala
Ala Glu Ile Arg Asp Pro Glu Lys Ala Ala Arg 20 25 30Val Trp Leu Gly
Thr Phe His Thr Ala Glu Asp Ala Ala Ile Ala Tyr 35 40 45Asp Glu Ala
Ala Leu Lys Phe Lys Gly Asn Lys Ala Lys Leu Asn Phe 50 55 60Pro Glu
Arg Val Gln Ser Thr Thr Asp Gln Phe Gly Ile Ser Tyr Leu65 70 75
80Ile Thr Asn Thr Asn His Gln Gln His Gln Phe Gln Pro Thr Asn Phe
85 90 95Leu Pro Asn Ser Asp 10065107PRTCucumis sativus 65Arg Val
Lys Arg Leu Lys Lys Asn Tyr Arg Gly Val Arg Gln Arg Pro1 5 10 15Trp
Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Ile Arg Ala Ala Arg 20 25
30Val Trp Leu Gly Thr Phe Asn Thr Ala Glu Asp Ala Ala Arg Ala Tyr
35 40 45Asp Glu Ala Ala Ile Lys Phe Arg Gly Pro Arg Ala Lys Leu Asn
Phe 50 55 60Pro Phe Pro Asp Tyr Ser Leu Ser Ser Thr Phe His Ser Ser
Pro Pro65 70 75 80Pro Ala Ser Thr Thr Thr Ser Ala Ser Ala Ser Phe
Ser Pro Ala Ala 85 90 95Pro Pro Pro Pro Pro Leu Leu Pro Thr Ser Thr
100 10566120PRTPopulus trichocarpa 66Met Ala Asp Ser Asp Asn Glu
Ser Gly Glu Gln Asn Asn Ser Asn Thr1 5 10 15Asn Tyr Ser Thr Glu Thr
Ser Pro Arg Glu Gln Asp Arg Leu Leu Pro 20 25 30Ile Ala Asn Val Ser
Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala 35 40 45Lys Ile Ser Lys
Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu 50 55 60Phe Ile Ser
Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu65 70 75 80Lys
Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr 85 90
95Leu Gly Phe Glu Asp Tyr Val Glu Pro Leu Lys Ile Tyr Leu Gln Lys
100 105 110Phe Arg Glu Met Glu Gly Glu Lys 115 12067116PRTSolanum
lycopersicum 67Met Ala Asp Ser Asp Asn Glu Ser Gly Gly His Asn Asn
Ala Asn Ser1 5 10 15Glu Gly Ser Thr Arg Glu Gln Asp Arg Phe Leu Pro
Ile Ala Asn Val 20 25 30Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn
Ala Lys Ile Ser Lys 35 40 45Asp Ala Lys Glu Thr Val Gln Glu Cys Val
Ser Glu Phe Ile Ser Phe 50 55 60Ile Thr Gly Glu Ala Ser Asp Lys Cys
Gln Arg Glu Lys Arg Lys Thr65 70 75 80Ile Asn Gly Asp Asp Leu Leu
Trp Ala Met Thr Thr Leu Gly Phe Glu 85 90 95Glu Tyr Val Glu Pro Leu
Lys Ile Tyr Leu Ala Lys Tyr Arg Glu Met 100 105 110Glu Gly Glu Lys
11568118PRTTheobroma cacao 68Met Ala Asp Ser Asp Asn Asp Ser Gly
Gly His Asn Asn Ser Asn Ala1 5 10 15Asn Asn Glu Leu Ser Pro Arg Glu
Gln Asp Arg Phe Leu Pro Ile Ala 20 25 30Asn Val Ser Arg Ile Met Lys
Lys Ala Leu Pro Ala Asn Ala Lys Ile 35 40 45Ser Lys Asp Ala Lys Glu
Thr Val Gln Glu Cys Val Ser Glu Phe Ile 50 55 60Ser Phe Ile Thr Gly
Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg65 70 75 80Lys Thr Ile
Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly 85 90 95Phe Glu
Asp Tyr Val Glu Pro Leu Lys Val Tyr Leu His Lys Phe Arg 100 105
110Glu Met Glu Gly Glu Arg 11569115PRTPanicum virgatum 69Met Pro
Asp Ser Asp Asn Glu Ser Gly Gly Pro Ser Asn Ala Glu Phe1 5 10 15Ser
Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25
30Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp
35 40 45Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe
Ile 50 55 60Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys
Thr Ile65 70 75 80Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu
Gly Phe Glu Asp 85 90 95Tyr Ile Glu Pro Leu Lys Leu Tyr Leu His Lys
Phe Arg Glu Leu Glu 100 105 110Gly Glu Lys 11570115PRTSetaria
italica 70Met Pro Asp Ser Asp Asn Glu Ser Gly Gly Pro Ser Asn Ala
Glu Phe1 5 10 15Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala
Asn Val Ser 20 25 30Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys
Ile Ser Lys Asp 35 40 45Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu
Phe Ile Ser Phe Ile 50 55 60Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg
Glu Lys Arg Lys Thr Ile65 70 75 80Asn Gly Asp Asp Leu Leu Trp Ala
Met Thr Thr Leu Gly Phe Glu Asp 85 90 95Tyr Ile Glu Pro Leu Lys Leu
Tyr Leu His Lys Phe Arg Glu Leu Glu 100 105 110Gly Glu Lys
11571115PRTSorghum bicolor 71Met Pro Asp Ser Asp Asn Glu Ser Gly
Gly Pro Ser Asn Ala Asp Phe1 5 10 15Ser Ser Pro Arg Glu Gln Asp Arg
Phe Leu Pro Ile Ala Asn Val Ser 20 25 30Arg Ile Met Lys Lys Ala Leu
Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45Ala Lys Glu Thr Val Gln
Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60Thr Gly Glu Ala Ser
Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile65 70 75 80Asn Gly Asp
Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp 85 90 95Tyr Ile
Glu Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu Glu 100 105
110Gly Glu Lys 11572115PRTZea mays 72Met Pro Asp Ser Asp Asn Glu
Ser Gly Gly Pro Ser Asn Ala Glu Phe1 5 10 15Ser Ser Pro Arg Glu Gln
Asp Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25 30Arg Ile Met Lys Lys
Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45Ala Lys Glu Thr
Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55 60Thr Gly Glu
Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile65 70 75 80Asn
Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp 85 90
95Tyr Val Glu Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu Leu Glu
100 105 110Gly Glu Lys 11573115PRTHordeum vulgare 73Met Pro Asp Ser
Asp Asn Asp Ser Gly Gly Pro Ser Asn Ala Asp Phe1 5 10 15Ser Ser Pro
Lys Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser 20 25 30Arg Ile
Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp 35 40 45Ala
Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Ile 50 55
60Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys Thr Ile65
70 75 80Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu
Asp 85 90 95Tyr Met Glu Pro Leu Lys Leu Tyr Leu His Lys Phe Arg Glu
Leu Glu 100 105 110Gly Glu Lys 11574118PRTOryza sativa 74Met Pro
Asp Ser Asp Asn Asp Ser Gly Gly Pro Ser Asn Tyr Ala Gly1 5 10 15Gly
Glu Leu Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala 20 25
30Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile
35 40 45Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe
Ile 50 55 60Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu
Lys Arg65 70 75 80Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met
Thr Thr Leu Gly 85 90 95Phe Glu Asp Tyr Val Asp Pro Leu Lys His Tyr
Leu His Lys Phe Arg 100 105 110Glu Ile Glu Gly Glu Arg
11575117PRTBrachypodium distachyon 75Met Pro Asp Ser Asp Asn Asp
Ser Gly Gly Pro Ser Asn Thr Gly Gly1 5 10 15Glu Leu Ser Ser Pro Arg
Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn 20 25 30Val Ser Arg Ile Met
Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser 35 40 45Lys Asp Ala Lys
Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser 50 55 60Phe Ile Thr
Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg Lys65 70 75 80Thr
Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe 85 90
95Glu Asp Tyr Val Asp Pro Leu Lys His Tyr Leu His Lys Phe Arg Glu
100 105 110Ile Glu Gly Glu Arg 11576115PRTTriticum aestivum 76Met
Pro Asp Ser Asp Asn Glu Asp Ser Gly Asn Ala Gly Gly Glu Leu1 5 10
15Ser Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro Ile Ala Asn Val Ser
20 25 30Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys
Asp 35 40 45Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu Phe Ile Ser
Phe Ile 50 55 60Thr Gly Glu Ala Ser Asp Lys Cys Gln Arg Glu Lys Arg
Lys Thr Ile65 70 75 80Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr
Leu Gly Phe Glu Asp 85 90 95Tyr Val Asp Pro Leu Lys His Tyr Leu His
Lys Phe Arg Glu Ile Glu 100 105 110Gly Glu Arg 11577118PRTGlycine
max 77Met Ala Asp Ser Asp Asn Asp Ser Gly Gly Ala His Asn Ala Gly
Lys1 5 10 15Gly Ser Glu Met Ser Pro Arg Glu Gln Asp Arg Phe Leu Pro
Ile Ala 20 25 30Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn
Ala Lys Ile 35 40 45Ser Lys Asp Ala Lys Glu Thr Val Gln Glu Cys Val
Ser Glu Phe Ile 50 55 60Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys
Gln Arg Glu Lys Arg65 70 75 80Lys Thr Ile Asn Gly Asp Asp Leu Leu
Trp Ala Met Thr Thr Leu Gly 85 90 95Phe Glu Asp Tyr Val Glu Pro Leu
Lys Gly Tyr Leu Gln Arg Phe Arg 100 105 110Glu Met Glu Gly Glu Lys
115
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